Supporting InformationGu et al. 10.1073/pnas.1525726113SI Materials and MethodsPrimary Neuron Culture.Rat hippocampi from day-18 embryos wereseeded on poly-L-lysine precoated 25-mm (for dual-TIRFM im-aging) or 18-mm (for immunocytochemistry) coverslips in Neuro-basal media (Invitrogen) containing 50 U/mL penicillin, 50 μg/mLstreptomycin, and 2 mM glutamax supplemented with 2% (vol/vol)B27 and 5% (vol/vol) FBS (plating medium). Media was replaced24 h after plating with feeding medium (plating medium withoutserum) and neurons were fed twice a week thereafter. Rat corticalneurons were prepared similarly and seeded on poly-L-lysine pre-coated 12-well plates for electroporation. Neurons were grown at37 °C and 5% (vol/vol) CO2/95% (vol/vol) air.

Transfection and Electroporation. Hippocampal neurons were trans-fected with Lipofectamine 2000 (Invitrogen) atDIV (day in vitro) 11–14 and were imaged or fixed 24–72 h posttransfection. Electro-poration of cortical neurons was performed at DIV 0 using AmaxaNucleofector kit (VPG-1003; Lonza) and Nucleofector device(Lonza). Three micrograms of pSuper-shRNAs were electroporatedinto 3 × 106 cortical neurons. The electroporated neurons wereseeded after electroporation and harvested 4 d postelectroporation.

shRNAs. To generate shRNAs targeting individual rat SNAPs andsyntaxins and VAMPs, pSuper was used as a vector for shRNAs.Oligos were annealed for direct subcloning into pSuper betweenBglII and HindIII sites (for scramble, SNAP23, SNAP29, andSNAP47 shRNAs) or between BglII and XhoI sites (for SNAP25,syntaxin1A, syntaxin1B, syntaxin4,VAMP1, andVAMP2 shRNAs).The shRNA sequences against SNAP23 and 47 were designedbased on siDESGN online tool (Dharmacon). The oligo se-quences targeting SNAP23 were 5′-GAT CCC CGG ATA TGGGCA ATG AAA TTT TCA AGA GAA ATT TCA TTG CCCATA TCC TTT TTA-3′ (sense) and 5′-AGC TTA AAA AGGATA TGG GCA ATG AAA TTT CTC TTG AAA ATT TCATTG CCC ATA TCC GGG-3′ (antisense). The oligo sequencestargeting SNAP47 were 5′-GAT CCC CAG GAA GAT GTTGAT GAT ATT TCA AGA GAA TAT CAT CAA CAT CTTCCT TTT TTA-3′ (sense) and 5′-AGC TTA AAA AAG GAAGAT GTT GAT GAT ATT CTC TTG AAA TAT CAT CAACAT CTT CCT GGG-3′ (antisense). The shRNA sequencesagainst SNAP29 were previously published by Pan et al. (31):5′ GAT CCC CGT GGA CAA GTT AGA TGT CAA TTTCAA GAG AAT TGA CAT CTA ACT TGT CCA CTT TTTA-3′ (sense) and 5′-AGC TTA AAA AGT GGA CAA GTTAGA TGT CAA TTC TCT TGA AAT TGA CAT CTA ACTTGT CCA CGG G-3′ (antisense). The oligo sequences targetingSNAP25, syntaxin1A, and syntaxin1B were obtained from ON-TARGET plus siRNAs of Dharmacon. The shRNA sequencesagainst SNAP25 (catalog no. J-093289-11) were 5′-GAT CCCCGG CTT CAT CCG CAG GGT AAT TCA AGA GAT TACCCT GCG GAT GAA GCC TTT TTC-3′ (sense) and 5′-TCGAGA AAA AGG CTT CAT CCG CAG GGT AAT CTC TTGAAT TAC CCT GCG GAT GAA GCC GGG-3′ (antisense).The shRNA sequences against syntaxin1A (catalog no. J-089172-11) were 5′-GAT CCC CCA CCA AAG GTC TCG GTA CATTCA AGA GAT GTA CCG AGA CCT TTG GTG TTT TTC-3′(sense) and 5′-TCG AGA AAA ACA CCA AAG GTC TCGGTA CAT CTC TTG AAT GTA CCG AGA CCT TTG GTGGGG-3′ (antisense). The shRNA sequence against syntaxin1B(catalog no. J-090348-12) were 5′-GAT CCC CCG GTC CAAGTT GAA AGC GAT TCA AGA GAT CGC TTT CAA CTTGGA CCG TTT TTC-3′ (sense) and 5′-TCG AGA AAA ACG

GTC CAA GTT GAA AGC GAT CTC TTG AAT CGC TTTCAA CTT GGA CCG GGG-3′ (antisense). The shRNA se-quences against syntaxin4 were designed based on the previ-ously published shRNA sequences targeting human syntaxin4(56): 5′-GAT CCC CAA GGA AGA AGC TGA TGA GAATTT CAA GAG AAT TCT CAT CAG CTT CTT CCT TTTTTT C-3′ (sense) and 5′-TCG AGA AAA AAA GGA AGAAGC TGA TGA GAA TTC TCT TGA AAT TCT CAT CAGCTT CTT CCT TGG G-3′ (antisense). The shRNA sequenceagainst VAMP1 (catalog no. J-091077-09) were 5′GAT CCC CGACCA GTA ACA GAC GAT TAT TCA AGA GAT AAT CGTCTG TTA CTG GTC TTT TTC 3′ (sense) and 5′ TCG AGAAAA AGA CCA GTA ACA GAC GAT TAT CTC TTG AATAAT CGT CTG TTA CTGGTCGGG 3′ (antisense). The shRNAsequences of VAMP2 (catalog no. J-090962-09) were 5′ GAT CCCCAC CAG AAG CTA TCG GAA CTT TCA AGA GAA GTTCCG ATA GCT TCT GGT TTT TTC 3′ (sense) and 5′ TCGAGA AAA AAC CAG AAG CTA TCG GAA CTT CTC TTGAAA GTT CCG ATA GCT TCT GGT GGG 3′ (antisense). Thescramble shRNA sequences are 5′-GAT CCC CGCGCGCTT TGTAGG ATT CGT TCA AGA GAC GAA TCC TAC AAA GCGCGC TTT TTA-3′ (sense) and 5′-AGC TTAAAAAGCGCGCTTTGT AGG ATT CGT CTC TTG AAC GAA TCC TAC AAAGCG CGC GGG-3′ (antisense).

Pharmacology. Chemicals and botulinum type A toxin were pur-chased from Sigma-Aldrich. TTX and dynasore were purchasedfrom Tocris Bioscience. Botulinum type B and C toxins werepurchased from METAbiologics.For Botox A treatment, Botox A was originally dissolved in a

solution containing 20 mM Hepes, 1.25% (wt/vol) lactose, and1 mg/mL BSA. Prior applying to neurons, Botox A was incubatedin ACSF containing 5 mM DTT for 30 min to activate the toxin;260 nM Botox A was applied to hippocampal neurons in mediafor 1 h and the cells were then imaged under TIRFM. The solventwas applied to the neurons in the control group.For Botox C treatment, 100 nM Botox B or 100 nM Botox C

was applied to hippocampal neurons in media for 6 h and thenimaged under TIRFM. The solvent (PBS) was applied in themedia of the control group.

Dual-TIRFM Imaging. An Olympus IX71 microscope with a plan-Apo objective (100×, N.A. 1.45, oil; Olympus) was used for dual-TIRFM imaging. The excitation laser was a 1-W Kr/Ar TIRFlaser with launch (SP_2018; Prairie Technologies). The fluorescentsignal of pHlourin was imaged with a 488-nm excitation laser(1.7 mW to the back aperture of the objective) and collectedthrough a 525- to 550-nm emission filter, and the fluorescentsignal of tdTomato was imaged with a 568-nm laser (2.8 mW tothe back aperture of the objective) and a 605- to 655-nm emissionfilter. Images were captured by a 512 × 512 back-illuminated CCDcamera with on-chip multiplication gain and 16- × 16-μm pixels(Cascade 512B; Photometrics) and MetaMorph acquisition soft-ware (Molecular Devices) at a rate of 1.4 Hz and 500-ms exposure(except some data in Fig. S1B: 100-ms exposure at 10 Hz). Neuronsbetween the ages of DIV 13 and 16 were used for imaging. Allimaging experiments were carried out in artificial cerebrospinal fluid[ACSF, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2,25 mM Hepes (pH 7.4), and 30 mM glucose]. The imaging tem-perature was maintained at 35–37 °C by a temperature controller(TC-202A; Harvard Apparatus). To increase the signal-to-noise

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ratio, we photobleached the fluorescent signals for 20 s withmaximal laser power under TIRF mode before data acquisition.

TIRF Data Analysis. Insertion kinetics of pHluorin-tagged receptorswere analyzed by custom-made automatic software. TIRF imagesof pH-GluA2 and pH-γ2S from a time-lapsed video were firstfiltered through a low-pass filter (57, 58) to enhance signal-to-noise ratio for the detection of event objects. Then a 2D Gaussiandistribution was used to fit the local intensity distribution of eachregional brightest pixel in the filtered image. A threshold value forpeak intensity and R-squared value of the fits were set manually.All of the image processing and analysis were performed usingcustom-made software developed in MATLAB.The distributions of pHluorin- or tdTomato-tagged receptor

insertions were analyzed by ImageJ software (National Institutesof Health). For each cell the image stacks from green and redchannels were properly aligned with fluorescent beads (Tetra-Speck Fluorescent Microspheres Size Kit; Molecular Probes). Wecropped a same rectangular region crossing the somatic area ingreen or red channels. Insertion events in the region were isolatedby subtraction of the sequential images 2–31 (acquired in 0.7–21.7 s) by the series of images from 1 to 30 (acquired in 0–21 s).Insertions were then counted by ImageJ under appropriatethresholds and normalized by the largest number of insertionsobtained along the axis. Normalized numbers of events wereplotted against the length of the soma. The total length of thesoma is normalized to 1.Insertion frequencies of pHluorin-tagged receptors were an-

alyzed by combining DiaTrack 3.03 software (Vallotton Semasopht),ImageJ (National Institutes of Health), and a custom-developedsoftware program, ImageSplice. Insertion events were generated bysubtraction of the sequential images 2–11 (acquired in 0.7–7.7 s) bythe series of images from 1 to 10 (acquired in 0–7 s) (ImageSplice).The number of insertions of each subtracted image from eachchannel is detected and counted by DiaTrack. The number of in-sertions was normalized to the surface area of the cell includingsomatic membrane and proximal dendritic membrane (ImageJ).

Single-Molecule Analysis. The numbers of pH-GluA2 or pH-γ2S inindividual exocytic vesicles were estimated as previously de-scribed (22). Insertions of pH-GluA2 and pH-γ2S in transfectedhippocampal neurons were imaged as described above. Un-der the same condition, fluorescent signals of purified EGFPmonomer absorbed to poly-L-lysine–coated coverslips were im-aged under TIRFM. Single molecules of EGFP were identifiedby the blinking and single step photobleaching characters. Theintensity of all of the fluorescent events, including pH-GluA2,pH-γ2S, and EGFP, were background-subtracted, plotted, andfitted by Gaussian curve (OriginPro 8; OriginLab). The intensitycorresponding to the peak of the curve was used to estimate thenumber of receptors in vesicles.

Live-Cell Imaging of Fluorescently Tagged Receptor Expression andPost-Golgi Trafficking.Expressions of EGFP-GluA2, tdt-GluA2,EGFP-γ2S, and tdt-γ2S in live neurons were imaged on a ZeissLSM510 using a 63× objective (N.A. 1.40) (Carl Zeiss MicroImagingGroup Inc.).To image post-Golgi trafficking in live neurons, neurons that

were seeded on coverslips were transfected with different com-binations of EGFP-GluA2, tdt-GluA2, EGFP-γ2S, and tdt-γ2Son DIV 11. The transfected neurons are incubated at 37 °C for2 h to allow protein expression. Cells were then transferred to25 mM Hepes (pH 7.4)-buffered Neurobasal medium (In-vitrogen) and incubated in 20 °C water bath for 5 h to allowEGFP- or tdt-tagged receptors to traffic to and accumulate in theGolgi apparatus. Cells were imaged and maintained at 32 °C usinga temperature controller (TC-202A; Harvard Apparatus) in ACSF;20 °C incubations and live-cell imaging were performed in the

presence of cycloheximide (20 μg/mL; Sigma). Live-cell imagingexperiments were performed on an Olympus IX71 microscope witha plan-Apo objective (100×, N.A. 1.45, oil; Olympus). The excitationlaser was a 1-W Kr/Ar TIRF laser with launch (SP_2018; PrairieTechnologies). The fluorescent signal of GFP was imaged with a488-nm excitation laser and collected through a 525- to 550-nmemission filter, and the fluorescent signal of tdTomato was imagedwith a 568-nm excitation laser and a 605- to 655-nm emission filter.To image intracellular trafficking of EGFP- or tdTomato-taggedreceptors, the angle of lasers was adjusted to epi-fluorescent mode.Images were captured by a 512 × 512 back-illuminated CCD camerawith on-chip multiplication gain and 16- × 16-μm pixels (Cascade512B; Photometrics) and MetaMorph acquisition software (Molec-ular Devices) at a rate of 1.4 Hz and 500-ms exposure.

Immunocytochemical Experiments. Surface expression of tdt-GluA2and tdt-γ2S (Fig. S3) was labeled by incubating live hippocampalneurons in ACSF containing 10% (wt/vol) BSA for 20 min at 10 °Cwith anti-DsRed antibody (Clontech), which also recognizestdTomato. Neurons were then washed twice with ice-cold ACSFcontaining 10% (wt/vol) BSA for washout of the excess anti-bodies and then fixed with 4% (vol/vol) paraformaldehyde and 4%(wt/vol) sucrose in ice-cold PBS at room temperature for 15 min.Alexa568-conjugated secondary antibodies (Invitrogen) were ap-plied following fixation to visualize the surface staining of DsRedantibody [in PBS containing 10% (wt/vol) BSA, room temperature,1 h]. Neurons were then permeabilized with PBS containing 0.25%Triton X-100 at room temperature for 20 min. Coverslips wereblocked in 10% (wt/vol) BSA in PBS at room temperature for 1 hand primary antibodies for synaptic marker proteins were thenapplied for 2 h: PSD95 (clone 6G6; Millipore), VGluT (Mil-lipore), gephyrin (Synaptic Systems), or VGAT (SynapticSystems). Alexa647-conjugated secondary antibodies (Invitrogen)were applied for 1 h to visualize the staining of these synapticmarkers. Total expressions of tdTomato-tagged receptors wererepresented by natural fluorescence signals of the receptors. En-dogenous surface γ2 was labeled following the similar protocolusing anti-γ2 N terminus antibody (Alomone Labs). Owing to thelow affinity of the antibody, endogenous surface GluA2 was labeledby incubating the anti-GluA2 N terminus antibody (MAB397;Millipore) with live neurons at room temperature for 30 min.To detect the expressions of tdt-GluA2, tdt-γ2S, endogenous

GluA1, and β2/3 (Fig. S2 B and E), dissociated hippocampalneurons were fixed and permeabilized as previously described.Expressions of tdt-GluA2 and tdt-γ2S were detected by anti-DsRed antibody. GluA1 was detected by anti-GluA1 N terminusantibody (clone 007, 4.9D; R.L.H. laboratory). β2/3 was detectedby anti-β2/3 N terminus antibody (Millipore). The accumulation ofEGFP-GluA2, tdt-GluA2, EGFP-γ2S, or tdt-γ2S in the Golgi ap-paratus after the 20 °C incubation was detected by staining ofEGFP-GluA2 and EGFP-γ2S with anti-GFP antibody (JH4030,made in laboratory), or staining of tdt-GluA2 and tdt-γ2S with anti-DsRed antibody. GM130 was stained with anti-GM130 antibody(BD Biosciences).All fluorescence images, including live neurons and fixed immu-

nostaining samples, were acquired on a Zeiss LSM510 using a 63×objective (N.A. 1.40) (Carl Zeiss MicroImaging Group Inc.). Fluo-rescent intensities were quantified using ImageJ (National Institutesof Health).

Electrophysiology. To examine functions of SNAP23 and 25 in syn-aptic transmission, spontaneous AMPA receptor-mediated mEPSCor GABA receptor-mediated mIPSC were measured in culturedhippocampal neurons on DIV 13–17, 3 d after transfection ofshRNAs specifically against SNARE23, SNARE25, or scramblecontrol. Neurons were perfused in Hepes-buffered extracellularsolution (143 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2,10 mM Hepes, 10 mM glucose, pH 7.2, osmolality 305–310 mOsm)

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in the presence of 1 μM TTX and 100 μM DL-2-amino-5-phospho-nopentanoic acid. Whole-cell recording pipettes (4–8 MOhm) werefilled with internal solution I (115 mM Cs-MeSO4, 0.4 mM EGTA,5 mM TEA-Cl, 2.8 mM NaCl, 20 mM Hepes, 3 mM Mg-ATP,0.5 mM Na2-GTP, pH 7.2, osmolality 295–300 mOsm) con-taining 100 μM picrotoxin for mEPSC recording or internalsolution II (120 mM CsCl, 11 mM EGTA, 5 mM TEA-Cl, 0.1 mMCaCl2, 20 mM Hepes, 3 mM Mg-ATP, 0.5 mM Na2-GTP) con-taining 10 μM NBQX for mIPSC recording. Cells were held at−70 mV holding potential and recording was performed at roomtemperature. Upon entering whole-cell mode, we allowed 5 minfor dialysis of the intracellular solution before collecting data.Signals were measured with a MultiClamp 700B amplifier anddigitized using a Digidata 1440A analog-to-digital board. Dataacquisition was performed with pClamp 10.2 software and digi-tized at 10 kHz. mEPSCs or mIPSCs were detected with a tem-plate matching algorithm in Clampfit 10.2 software. All equipmentand software are from Axon Instruments/Molecular Devices.Averaged mEPSC and mIPSC amplitude and frequency werecalculated by collecting 2–4 min of recording for at least 100events for each cell. All data are presented as mean ± SEM.Statistical significance was calculated using a Mann–Whitney test.

Western Blot and Antibodies. Cortical neurons were harvested 4 dafter electroporation (DIV 4) in 60 μL SDS lysis buffer (1% SDS,1 mM EDTA, and protease inhibitor mixture) and further di-luted in 300 μL dilution buffer (1% TritonX-100, 1 mM EDTA,1 mM EGTA, and protease inhibitor mixture). Endogenous ex-pression of SNAP23, SNAP29, SNAP47, syntaxin1A, syntaxin1B,syntaxin4, and VAMP2 was detected by antibodies specific to eachprotein (Synaptic Systems). Endogenous expression of SNAP25was detected by anti-SNAP25 antibody (clone SMI-81R; Covance).Endogenous expression of VAMP1 was detected by anti-VAMP1antibody (Novus).

Subcellular Fractionation. Cerebral cortex from one adult maleSprague–Dawley rat (∼P60) was homogenized and fractionatedas previously described (44). The brain was weighed and ho-mogenized in 10 volumes of homogenization buffer (0.32 M su-crose, 4 mM Hepes, pH 7.4, and protease inhibitors) by 30 strokesin a Teflon-glass homogenizer (whole-brain homogenate, H). TheH fraction was centrifuged for 10 min at 1,100 × g to remove celldebris and nuclei (pellet 1, P1). The supernatant (S1) was centri-fuged for 10 min at 9,200 × g and the pellet (P2) was removed.Supernatant from this centrifugation (S2) was further centrifugedfor 2 h to obtain the microsomal pellet (P3) and soluble fraction(S3). Each fraction was normalized based on their protein con-centrations before being analyzed by Western blot.

ElectronMicroscopy.The morphological characterization of the P3fraction was done as previously described (6). The P3 pellet wasfixed in 5% (vol/vol) paraformaldehyde (freshly prepared fromEM-grade prill form) + 0.1% glutaraldehyde (EM grade) + 0.1%tannic acid + 3 mM MgCl2 + 0.1 M phosphate buffer (pH 7.2–7.4; Sorensen’s) at 4 °C overnight under gentle agitation. Fol-lowing rinsing with 3% (wt/vol) sucrose + 3 mM MgCl2 + 0.1 Mphosphate buffer, samples were postfixed with 1% osmium te-troxide (nonreduced) + 3 mM MgCl2 + 0.1 M phosphate bufferfor 1 h and then rinsed with 3% (wt/vol) sucrose + 0.1 Mphosphate buffer. The samples were immersed for 30 min withfiltered 2% (wt/vol) uranyl acetate (aqueous) and dehydratedthrough a graded series of ethanol from 50 to 100% (vol/vol).Dehydrated samples were transferred through propylene oxideand embedded in Eponate 12 (Pella) and cured at 60 °C for 2 d.Thin sections were cut on a Riechert Ultracut E with a Dia-tome Diamond knife. Eighty-nanometer sections were picked upon formvar-coated 1- × 2-mm copper slot grids (Pella) and stainedwith uranyl acetate followed by lead citrate.

For the postembed immunoelectron microscopy, P3 sampleswere fixed in 4% (vol/vol) paraformaldehyde + 0.2% gluta-raldeyde + 0.1 M Na3PO4 buffer + 3 mM MgCl2 overnight at4 °C. Fixation and all subsequent steps up to infiltration werecarried out at 4 °C followed by curing at 50 °C. After rinsing with0.1 M phosphate + 3% (wt/vol) sucrose, samples were incubatedwith 0.12% tannic acid (Malinkrodt) for 1 h. Upon buffer rinse,uncross-linked glutaraldeyde was reduced for 30 min with50 mM NH4Cl in buffer. After a 45-min rinsing in 0.1 M maleatebuffer (pH 6.2), fractions were en bloc-stained in 2% (wt/vol)uranyl acetate (filtered) in maleate for 30 min. After ethanol se-ries dehydration, samples were infiltrated with a 90% (vol/vol)ethanol: LR White (with catalyst) (2:1) mixture for 1 h. Sampleswere further infiltrated in 90% (vol/vol) ethanol:LR White (1:1and 1:3) then placed in pure LR White overnight at 4 °C . Afterthree changes in fresh LR White (4 °C), samples were polymer-ized in tightly sealed gelatin capsules (00) at 50 °C for 24 h. Po-lymerized blocks were trimmed and sectioned as described above.For immunolabeling grids were treated with 5% (wt/vol) sodium pe-riodate (aqueous; Sigma) followed by 50 mMNH4Cl in TBS (50 mMTrizma base + 150 mMNaCl, pH 7.4). After a block in 10% (vol/vol)normal goat serum (NGS) + 10% (vol/vol) donkey serum (DS) inTBST (10 mM Tris + 500 mMNaCl + 0.05% Tween 20, pH 7.2) for10 min, grids were incubated with primary antibodies, including anti-GluA2 N terminus antibody (Millipore) at 1:5 and anti-γ2 N termi-nus antibody (Synaptic Systems) at 1:16–24, in 1% NGS + 1% DS inTBST overnight at 4 °C. Samples without the first antibodies servedas controls. Grids were then incubated for 10 min in 1% BSA inTBST and sequentially incubated in gold-conjugated secondary an-tibodies (6 nm Au-goat anti-mouse, 1:40, and 12 nm Au-donkey anti-rabbit, 1:40; Jackson Laboratories) at room temperature for 1 h foreach antibody. After rinsing in TBS then in distilled H2O grids wereincubated in 2% (vol/vol) glutaraldehyde for 5 min. After rinsinggrids were stained with 2% (wt/vol) filtered uranyl acetate (aqueous)and then allowed to dry.All grids were viewed with a Phillips CM 120 TEM operating at

80 kV and images were captured with anXR 80-8Megapixel CCDcamera by AMT.

Quantitation of Immunogold Labeling. The areas of the sectionswith immunogold labeling were photographed randomly at finalmagnification of 65,000×. Numbers of 50- to 300-nm diametervesicles, which were unlabeled, or labeled by 6-nm or 12-nm im-munogold particles, were counted manually. Evaluation of statisti-cal significance of colabeling of GluA2 and γ2 was done to test thenull hypothesis that GluA2-containing vesicles and γ2-containingvesicles are independent vesicle populations. The observed fre-quencies (Oi) of four vesicle populations, including unlabeled, la-beled for GluA2 only, labeled for γ2 only, and labeled for bothGluA2 and γ2, were compared with the frequencies expected (Ei) ifGluA2 and γ2 were distributed independently. The deviation of theobserved from the expected frequencies was quantified by Pearson’sstatistic (χ2 test),

χ2 =X4

i=1

ðOi −EiÞ2Ei

.

The corresponding P value was obtained from a χ2 distributionwith one degree of freedom. The distribution of the observedvesicle populations is not significantly different from the nullhypothesis when two-sided P > 0.05.

Statistics. Student t test, Mann–Whitney test, or one-way ANOVAwas used for statistical analysis and was performed using Prismsoftware (GraphPad Software). P < 0.05 was taken as a statisticallysignificant difference, unless otherwise noted. Values are reportedas mean ± SEM, unless otherwise noted.

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Fig. S1. Characterization of surface dynamic events of pH-GluA2 and pH- γ2S. (A) Duration of pH-GluA2 events is longer than that of pH-γ2S events. Each datapoint represents one cell. (B) The frequency of dynamic TIRF events of pH-γ2S is significantly higher than that of pH-GluA2 under two different imagingfrequencies. The exposure time was 100 ms for 10-Hz imaging. The exposure time was 500 ms for 1.4-Hz imaging. Each data point represents one cell.(C) Frequencies of dynamic TIRF events of pH-GluA2 and pH-γ2S are sensitive to pH of extracellular solution, which is indicated in the figure. Fifty millimolarNH4Cl was used in the extracellular solution to alkalinize all of the intracellular vesicles. (D) Frequency of the dynamic events of GluA2 and γ2S is not affectedby neuronal activity. One micromolar TTX or 12 mM KCl were applied to hippocampal neurons in the media for 1 h and the cells were then imaged underTIRFM. Asterisk indicates statistical significance. n.s., no statistical significance. (E , Top) Sequential images showing the kinetics of surface-absorbed EGFPmonomer under single-step photobleaching visualized by TIRFM. Image interval: 0.7 s. (Scale bar: 1 μm.) (Bottom) An example of fluorescent intensity trace ofEGFP monomer under single-step photobleaching. (F) Binned fluorescence intensity distribution of individual EGFP monomers (gray bars) and fitted Gaussiancurve based on the distribution (purple curve). The dashed line shows the peak intensity of the Gaussian curve. n = 561. (G) Binned fluorescence intensitydistribution of individual pH-GluA2–containing vesicles (gray bars) and fitted Gaussian curve based on the distribution (purple curve). The dashed line showsthe peak intensity of the Gaussian curve. n = 446. (H) Binned fluorescence intensity distribution of individual pH-γ2S–containing vesicles (gray bars) and fittedGaussian curve based on the distribution (purple curve). The dashed line shows the peak intensity of the Gaussian curve. n = 733.

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Fig. S2. Characterization of tdTomato-GluA2 and tdTomato-γ2S. (A) Coexpression of EGFP-GluA2 (top left) and tdt-GluA2 (top right) in live hippocampalneurons. (Scale bar: 20 μm.) High-magnification images from the selected region are shown in bottom panels: bottom left, EGFP-GluA2; bottom middle, tdt-GluA2; bottom right, overlay left and middle panels. (Scale bar: 5 μm.) (B) Colocalization of tdt-GluA2 and endogenous GluA1 in hippocampal neurons at DIV19. Top left, total tdt-GluA2 stained with anti-DsRed antibody; top middle, endogenous GluA1 stained with anti-GluA1 antibody; top right, overlay of total tdt-GluA2 and endogenous GluA1. (Scale bar: 20 μm.) High-magnification images from the selected region are shown in bottom panels. (Scale bar: 5 μm.)(C) Quantification of colocalization of tdt-GluA2 and endogenous GluA1 in B. Magenta column: 43.4 ± 4.6% of total tdt-GluA2 colocalizes with total GluA1.Green column: 75.5 ± 2.2% of synaptic tdt-GluA2 colocalizes with synaptic GluA1. (D) Similar to Awith coexpressions of EGFP-γ2S and tdt-γ2S. (E) Colocalizationof tdt-γ2S and endogenous β2/3 in hippocampal neurons at DIV 19. Top left, total tdt-γ2S stained with anti-DsRed antibody; top middle, endogenous β2/3stained with anti-β2/3 antibody; top right, overlay of total tdt-γ2S and endogenous β2/3. (Scale bar: 20 μm.) High-magnification images from the selectedregion are shown accordingly in bottom panels. (Scale bar: 5 μm.) (F) Quantification of colocalization of tdt-γ2S and endogenous β2/3 in E. Magenta column:51.3 ± 4.4% of total tdt-γ2S colocalizes with total β2/3. Green column: 85.4 ± 1.4% of synaptic tdt-γ2S colocalizes with synaptic β2/3. Each group of quantifieddata is collected from three cells and three different areas were used in each cell.

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Fig. S3. Colocalizations of tdt-GluA2 with PSD95 and VGluT. (A) Immunostainings of surface tdt-GluA2 and PSD95 in hippocampal neurons at DIV 18. Top left,fluorescent signal of total tdt-GluA2 without staining; top middle, surface tdt-GluA2; top right, endogenous PSD95. (Scale bar: 20 μm.) High-magnificationimages from the selected region are shown in middle panels. (Scale bar: 5 μm.) Bottom panels: overlaid figures of middle figures as indicated in the figure.(B) Quantification of colocalization of tdt-GluA2 with PSD95 in A. Magenta column: 37.3 ± 3.1% of total tdt-GluA2 colocalizes with total PSD95. Green column:31.4 ± 2.6% of surface tdt-GluA2 colocalizes with total PSD95. Stripped magenta column: 82.1 ± 2.0% of synaptic total tdt-GluA2 colocalizes with synapticPSD95. Stripped green column: 69.4 ± 3.0% of synaptic surface tdt-GluA2 colocalizes with synaptic PSD95. (C) Immunostainings of surface tdt-GluA2 and VGluTin hippocampal neurons at DIV 18. Top left, fluorescent signal of total tdt-GluA2 without staining; top middle, surface tdt-GluA2; top right, endogenous VGluT.(Scale bar: 20 μm.) High-magnification images from the selected region are shown in middle panels. (Scale bar: 5 μm.) Bottom panels: overlaid figures of middlefigures as indicated in the figure. (D) Quantification of colocalization of tdt-GluA2 with VGluT in C. Magenta column: 18.7 ± 2.8% of total tdt-GluA2 colocalizeswith total VGluT. Green column: 18.3 ± 2.0% of surface tdt-GluA2 colocalizes with total VGluT. Stripped magenta column: 46.9 ± 3.1% of synaptic total tdt-GluA2 colocalizes with synaptic VGluT. Stripped green column: 42.9 ± 2.0% of synaptic surface tdt-GluA2 colocalizes with synaptic VGluT. Each group ofquantified data is collected from three cells and three different areas were used in each cell.

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Fig. S4. Colocalizations of tdt-γ2S with gephyrin and VGAT. (A) Immunostainings of surface tdt-γ2S and gephyrin in hippocampal neurons at DIV 16. Top left,fluorescent signal of total tdt-γ2S without staining; top middle, surface tdt-γ2S; top right, endogenous gephyrin. (Scale bar: 20 μm.) High-magnification imagesfrom the selected region are shown in middle panels. (Scale bar: 5 μm.) Bottom panels: overlaid figures of middle figures as indicated in the figure.(B) Quantification of colocalization of tdt-γ2S with gephyrin in E. Magenta column: 65.0 ± 6.0% of total tdt-γ2S colocalizes with total gephyrin. Green column:23.8 ± 3.3% of surface tdt-γ2S colocalizes with total gephyrin. Stripped magenta column: 84.6 ± 2.0% of synaptic total tdt-γ2S colocalizes with synaptic ge-phyrin. Stripped green column: 62.8 ± 3.7% of synaptic surface tdt-γ2S colocalizes with synaptic gephyrin. (C) Immunostainings of surface tdt-γ2S and VGAT inhippocampal neurons at DIV 16. Top left, fluorescent signal of total tdt-γ2S without staining; top middle, surface tdt-γ2S; top right, endogenous VGAT. (Scalebar: 20 μm.) High-magnification images from the selected region are shown in middle panels. (Scale bar: 5 μm.) Bottom panels: overlaid figures of middlefigures as indicated in the figure. (D) Quantification of colocalization of tdt-γ2S with VGAT in E. Magenta column: 56.6 ± 7.1% of total tdt-γ2S colocalizes withtotal VGAT. Green column: 25.9 ± 3.8% of surface tdt-γ2S colocalizes with total VGAT. Stripped magenta column: 77.9 ± 1.8% of synaptic total tdt-γ2S co-localizes with synaptic VGAT. Stripped green column: 63.1 ± 4.2% of synaptic surface tdt-γ2S colocalizes with synaptic VGAT. Each group of quantified data iscollected from three cells and three different areas were used in each cell.

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Fig. S5. Cleavage of SNARE proteins by botulinum toxins. (A) Endogenous SNAP25 was cleaved by Botox A. Botox A (260 nM) was applied to hippocampalneurons in media for 1 h. The cells were then lysed and the endogenous SNAP25 level was analyzed. (B) Quantification of cleavage of SNAP25 by Botox A.Averaged protein levels of SNAP25 were normalized by the control without toxin treatment. (C) Endogenous VAMP2 was cleaved by Botox B. Botox B (100 nM)was applied to hippocampal neurons in media for 6 h. The cells were then lysed and the endogenous VAMP2 level was analyzed. (D) Quantification of cleavageof VAMP2 by Botox B. Averaged protein levels of VAMP2 were normalized by the control without toxin treatment. (E) Endogenous syntaxin1A, 1B, 2, 3, andSNAP25 were cleaved by Botox C. Botox C (100 nM) was applied to hippocampal neurons in media for 6 h. The cells were then lysed and the endogenousSNAP25, syntaxin 1A, 1B, 2, and 3 levels were analyzed. (F–J) Quantification of cleavage of syntaxin1A, 1B, 2, 3, and SNAP25 by Botox C. Averaged protein levelsof each SNARE were normalized by the control without toxin treatment. Quantified data shown in B, D, and F–J are from three independent experiments.Asterisk indicates statistical significance.

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Fig. S6. Lateral diffusion of pH-GluA2 or pH-γ2S following exocytosis. (A) Dynamics of an exocytic event of pH-GluA2. Note that after exocytosis, one exocyticevent splits into two events, which separately diffuse away from the insertion spot. (B) Fluorescence time courses on the right panel represent averagedfluorescence changes of the pH-GluA2 event in A in selected regions shown on the left. The green trace represents the fluorescence time course in the center ofthe exocytic spot (green circle). The magenta trace represents the fluorescence change over time in the annulus surrounding the exocytic spot (the areabetween green and magenta circles). The baseline was subtracted in each trace. The maximal fluorescence was normalized as 1. (C) Averaged fluorescence timecourse of multiple pH-GluA2 insertion events on the exocytic spot (green) and surrounding region (magenta). Note that the magenta trace peaks when the greentrace starts decaying. n = 30. (D) Peak time of fluorescence of pH-GluA2 events on the exocytic spot and surrounding region. n = 30. (E) Dynamics of an exocyticevent of pH-γ2S. (F) Fluorescence time courses on the right panel represent averaged fluorescence changes of the pH-γ2S event in E in selected regions, as indicatedon the left. (G) Averaged fluorescence time course of multiple pH-γ2S insertion events on the exocytic spot (green) and surrounding region (magenta). n = 30. (H)Peak time of fluorescence of pH-γ2S events on the exocytic spot and surrounding region. n = 30. Asterisk indicates statistical significance.

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Fig. S7. shRNA knockdown of SNARE proteins, and effects of shRNAs of SNARE proteins on exocytosis of GluA2 and γ2S. (A, B, and D–I) shRNA knockdown ofSNARE proteins. Three micrograms of shRNA specifically targeting each SNARE protein was electroporated into dissociated rat cortical neurons at DIV 0 im-mediately before plating. The cells were lysed 4 days after electroporation and expressions of endogenous SNARE proteins were analyzed. Tubulin was used asan internal control in all experiments. (A) SNAP23; (B) SNAP25; (D) SNAP29; (E) SNAP47; (F) syntaxin1A; (G) syntaxin1B; (H) syntaxin4; (I) VAMP1 and VAMP2.(C) Specificity of SNAP23 and SNAP25 shRNAs. shRNA specificity was tested in HEK293T cells. Myc-SNAP23 and GFP-SNAP25 were cotransfected with eitherSNAP23 or SNAP25 shRNA. Two days after transfection cells were lysed and expression of myc-SNAP23 and GFP-SNAP25 was determined by blotting with anti-myc and anti-GFP antibodies, respectively. (J and K) SNAP25 is required for both somatic (J) and dendritic (K) exocytosis of GluA2, but not γ2S. Scramble shRNA(Scr), SNAP25 shRNA, shRNA-resistant SNAP25 (25), or SNAP23 (23) were coexpressed with pH-GluA2 or pH-γ2S in hippocampal neurons. One day aftertransfection, exocytosis of pH-GluA2 or pH-γ2S were imaged under TIRFM. The somatic and dendritic exocytic events were then analyzed separately. (L and M)SNAP23 is required for both somatic (L) and dendritic (M) exocytosis of γ2S, but not GluA2. Scramble shRNA (Scr), SNAP23 shRNA, shRNA-resistant SNAP23 (23),or SNAP25 (25) were coexpressed with pH-GluA2 or pH-γ2S in hippocampal neurons. Two days after transfection, exocytosis of pH-GluA2 or pH-γ2S wereimaged under TIRFM. The somatic and dendritic exocytic events were then analyzed separately. (N) SNAP29 is not required for exocytosis of GluA2 and γ2S. Indissociated hippocampal neurons, scramble shRNA (Scr) or SNAP29 shRNA (29) was coexpressed with pH-GluA2 or pH-γ2S. Two days after transfection exo-cytosis of pH-GluA2 or pH-γ2S were imaged under TIRFM. (O) SNAP47 is not required for exocytosis of GluA2 and γ2S. In dissociated hippocampal neurons,scramble shRNA (Scr) or SNAP47 shRNA (47) was coexpressed with pH-GluA2 or pH-γ2S. Two days after transfection exocytosis of pH-GluA2 or pH-γ2S wereimaged under TIRFM. (P) Syntaxin4 is not required for exocytosis of GluA2 and γ2S. In dissociated hippocampal neurons, scramble shRNA (Scr) or syntaxin4shRNA (STX4) was coexpressed with pH-GluA2 or pH-γ2S. Two days after transfection exocytosis of pH-GluA2 or pH-γ2S were imaged under TIRFM. (Q) VAMP1is not required for exocytosis of GluA2 and γ2S. In dissociated hippocampal neurons, scramble shRNA (Scr) or VAMP1 shRNA was coexpressed with pH-GluA2 orpH-γ2S. Three days after transfection exocytosis of pH-GluA2 or pH-γ2S were imaged under TIRFM. Asterisks indicate statistical significances. n.s., no statisticalsignificance.

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Fig. S8. Knockdown of SNAP23 and SNAP25 does not affect surface expression of endogenous GluA2 and γ2, respectively. (A) Knockdown of SNAP23 did notaffect surface expression of endogenous GluA2. In dissociated hippocampal neurons, scramble shRNA, SNAP25 shRNA, or SNAP23 shRNA were coexpressedwith EGFP to label transfected cells. Surface levels of endogenous GluA2 were stained 72 h after transfection. Right panels show higher-magnification imagesof the individual processes boxed in left panels. N, surface GluA2 in nontransfected neurons; T, surface GluA2 in transfected neurons. (Scale bar: 20 μm.)(B) Quantification of endogenous surface GluA2 levels in A. Relative GluA2 surface level of transfected neurons is represented by a ratio of surface GluA2intensity in transfected cells to that of surrounding nontransfected cells. The ratios were further normalized by the average of the control sample transfectedwith the scramble shRNA. Scr: scramble shRNA. 23: shRNA-resistant SNAP23. 25: SNAP25. n = 18–22 neurons for each group. Five processes were selected fromeach neuron. (C) Knockdown of SNAP25 did not affect surface expression of GABAA receptor γ2 subunit. In dissociated hippocampal neurons at DIV 11,scramble shRNA, SNAP23 shRNA, or SNAP25 shRNA were coexpressed with EGFP to label transfected cells. Seventy-two hours after the transfection, endog-enous γ2 on the surface was stained. Right panels show higher-magnification images of individual processes boxed in left panels. N, surface γ2 in non-transfected cells; T, surface γ2 in cells transfected with shRNAs. (D) Quantification of the surface γ2 levels in C. Relative γ2 surface level is represented by a ratioof surface γ2 intensity in transfected cells to that of surrounding nontransfected cells. The ratios were further normalized by the average of the control sampletransfected with the scramble shRNA. Scr: scramble shRNA. 25: SNAP25 shRNA. 23: SNAP23 shRNA. n = 25–30 neurons for each group. Five processes wereselected from each neuron. Asterisks indicate statistical significances. n.s., no statistical significance.

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Fig. S9. Distributions of exocytosis of AMPA and GABAA receptors on the plasma membrane under coexpression of GluA1 or GluA3. (A) Demonstration ofevent detection by automated program. A raw image of pH-GluA2 from TIRF imaging was first obtained (Left). Event enhancement filtering was applied toreduce the noise (Middle). Objects with high intensity were then identified as events and their locations in the image were calculated (Right). (B) Averageddistribution of insertion events of pH-γ2S and tdt-GluA2 with coexpression of GluA1. The green and magenta curves represent the exocytic event distributionsof pH-γ2S and tdt-GluA2, respectively. n = 17. (C) Averaged distribution of insertion events of pH-GluA2 and tdt-γ2S with coexpression of GluA1. The green andmagenta curves represent the exocytic event distributions of pH-GluA2 and tdt-γ2S, respectively. n = 16. (D) Averaged distribution of insertion events of pH-γ2Sand tdt-GluA2 with coexpression of GluA3. The green and magenta curves represent the exocytic event distributions of pH-γ2S and tdt-GluA2, respectively. n =12. (E) Averaged distribution of the insertion events of pH-GluA2 and tdt-γ2S with coexpression of GluA3. The green and magenta curves represent theexocytic event distributions of pH-GluA2 and tdt-γ2S, respectively. n = 13. Asterisk indicates statistical significance.

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Fig. S10. The trafficking of EGFP- or tdTomato-tagged GluA2 and γ2S was blocked in Golgi apparatus after 20 °C incubation, and cotrafficking of GluA2 or γ2Swith different fluorescent tags when they exit the Golgi apparatus. (A) EGFP-γ2S, tdt-γ2S, EGFP-GluA2, or tdt-GluA2 were expressed in hippocampal neurons atDIV 11. After the incubation at 20 °C, EGFP-γ2S, tdt-γ2S, EGFP-GluA2, or tdt-GluA2 were stained by anti-GFP or tdTomato antibodies with a costaining of Golgimarker GM130. In the overlaid images, magenta shows the GM130 staining and green shows the staining of EGFP- or tdTomato-tagged receptors. (Scale bar:20 μm.) (B) Time series of an example of a post-Golgi trafficking vesicle containing EGFP-γ2S and tdt-γ2S, as indicated by arrows (green, magenta, and white) atcorresponding locations. For each time point, top: time-series images of EGFP-γ2S; middle: time-series images of tdt-γ2S; bottom: overlaid time-series images oftop and middle panels. (Scale bar: 2.5 μm.) See Movie S6 for complete time lapse. The kymographs show the trafficking of the vesicle along its trajectory forEGFP-γ2S, tdt-γ2S, and overlaid signal. (C) Similar to B, time series of an example of a post-Golgi trafficking vesicle containing EGFP-GluA2 and tdt- GluA2, asindicated by arrows (green, magenta, and white) at corresponding locations. See Movie S6 for complete time lapse.

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Movie S1. Plasma membrane insertions of pH-GluA2. pH-GluA2 was expressed in hippocampal neurons. Two days after transfection, plasma membraneinsertions of pH-GluA2 under TIRFM with an excitation laser at 488 nm. The movie was taken at 500-ms exposure and 1.4-Hz acquisition frequency.

Movie S1

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Movie S2. Plasma membrane insertions of pH-γ2S. pH-γ2S was expressed in hippocampal neurons. Two days after transfection, plasma membrane insertionsof pH-GluA2 under TIRFM with an excitation laser at 488 nm. The movie was taken at 500-ms exposure and 1.4-Hz acquisition frequency.

Movie S2

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Movie S3. Identification of insertion events of pH-GluA2 using automatic program. Insertion events of pH-GluA2 were automatically identified by a custom-made program under a proper threshold. The events were marked as magenta dots.

Movie S3

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Movie S4. Identification of insertion events of pH-γ2S using automatic program. Insertion events of pH-γ2S were automatically identified by a custom-madeprogram under a proper threshold. The events were marked as magenta dots.

Movie S4

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Movie S5. Plasma membrane insertions of tdt-GluA2 and pH-γ2S have different distributions. tdt-GluA2 and pH-γ2S were coexpressed in hippocampalneurons. Two days after transfection, plasma membrane insertions of tdt-GluA2 and pH-γ2S were simultaneously imaged under dual TIRFM. Left channelrepresents insertions of pH-γ2S, which excited by a laser at 488 nm. Right channel represents insertions of tdt-GluA2, which excited by a laser at 568 nm. Themovie was taken at 500-ms exposure and 1.4-Hz acquisition frequency.

Movie S5

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Movie S6. Cotrafficking of EGFP-γ2S/tdt-γ2S and EGFP-GluA2/tdt-GluA2 after exiting the Golgi apparatus. Trafficking vesicles were imaged at 32 °C at 0.7-sinterval after a preincubation at 20 °C. Top panel: cotrafficking of EGFP-γ2S and tdt-γ2S. Left channel represents a trafficking of EGFP-γ2S-containing vesicle.Middle channel represents the trafficking of the same vesicle containing tdt-γ2S. Right channel shows an overlaid movie of left and middle channels, rep-resenting a cotrafficking vesicle containing both EGFP-γ2S (green) and tdt-γ2S (magenta). Arrows point out the position of EGFP-γ2S/tdt-γ2S vesicle in eachchannel. Bottom panel: cotrafficking of EGFP-GluA2 and tdt-GluA2. Left channel represents the trafficking of an EGFP-GluA2-containg vesicle. Middle channelrepresents the trafficking of the same vesicle containing tdt-GluA2. Right channel shows an overlaid movie of left and middle channels, representing a co-trafficking vesicle containing both EGFP-GluA2 (green) and tdt-GluA2 (magenta). Arrows point out the position of EGFP-GluA2/tdt-GluA2 vesicle in eachchannel. (Scale bar: 2.5 μm.)

Movie S6

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Movie S7. Trafficking of EGFP-GluA2 and tdt-γ2S after exiting the Golgi apparatus. Trafficking vesicles containing EGFP-GluA2 or tdt-γ2S were simultaneouslyimaged at 32 °C at 0.7-s interval after a preincubation at 20 °C. Left channel represents the EGFP-GluA2 channel. Middle channel represents the tdt-γ2Schannel. Right channel shows an overlaid movie of left and middle channels. EGFP-GluA2 is shown in green and tdt-γ2S is shown in magenta. Top panel: anexample of a trafficking vesicle containing only tdt-γ2S, but not EGFP-GluA2. Arrows point out the position of tdt-γ2S-vesicle in each channel. Bottom panel: anexample of a trafficking vesicle containing only EGFP-GluA2, but not tdt-γ2S. Arrows point out the position of EGFP-GluA2-vesicle in each channel. (Scale bar:2.5 μm.)

Movie S7

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Movie S8. Trafficking of EGFP-γ2S and tdt-GluA2 after exiting the Golgi apparatus. Trafficking vesicles containing EGFP-γ2S or tdt-GluA2 were simultaneouslyimaged at 32 °C at 0.7-s interval after a preincubation at 20 °C. Left channel represents the EGFP-γ2S channel. Middle channel represents the tdt-GluA2channel. Right channel shows an overlaid movie of left and middle channels. EGFP-γ2S is shown in green and tdt-GluA2 is shown in magenta. Top panel: anexample of a trafficking vesicle containing only EGFP-γ2S, but not tdt-GluA2. Arrows point out the position of EGFP-γ2S-vesicle in each channel. Bottom panel:an example of a trafficking vesicle containing only tdt-GluA2, but not EGFP-γ2S. Arrows point out the position of tdt-GluA2-vesicle in each channel. (Scale bar:2.5 μm.)

Movie S8

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