Supporting InformationLi et al. 10.1073/pnas.1601918113SI Materials and MethodsAntibodies and Reagents. Affinity-purified rabbit antibodies againsthuman BIG1 and BIG2 have been described (23). Antibodiesagainst β-catenin (610154), α-catenin (610194), syntaxin-6 (610635),calreticulin (612137), and GM130 (610823) were purchased fromBD Biosciences; against HA (3724), phospho-(S675) β-catenin(4176), nonphosphorylated (S33/37/T41) β-catenin (8814), LRP6(2560), and Frizzled 6 (5158) from Cell Signaling; against activeβ-catenin (05-665), β-catenin (06-734), and GAPDH (AB2302)from Millipore; against phospho-(Y654) β-catenin (CP4021) fromECM Biosciences; against PKA catalytic subunit α (sc-28315) andN-cadherin (sc-7939) from Santa Cruz Biotechnology; againstFLAG (F1804), c-myc (C3956), α-tubulin (T5168), β-actin (A5441),and βCOP (G6160) from Sigma-Aldrich; and against GFP (632592)from Clontech. Horseradish peroxidase-conjugated sheep anti-rabbit and anti-mouse Ig (IgG) and TnT T7 coupled wheat germextract system were purchased from Promega and Alexa Fluor 594-or 488-conjugated goat anti-rabbit or anti-mouse IgG (H+L) fromInvitrogen. Protease inhibitor mixture tablets were purchased fromRoche, Wnt3a from R&D Systems, and inhibitors of PLD1 andPLD2 (5-fluoro-2-indolyl des-chlorohalopemide, FIPI) and of PKA(H89) from Sigma-Aldrich.

Expression Plasmids.Plasmids β-catenin-pcDNA3 (67), GSK3β-pCMV-Tag 5A (68), M50 Super 8× TOP-Flash, and M51 Super 8× FOP-Flash (69) were purchased from Addgene and those encodingRenilla (pRL-TK) from Promega. For yeast two-hybrid analyses,GSK3β and fragments of BIG1 and BIG2 were subcloned intopGADT7, and β-catenin into pGBKT7 vectors. Plasmids pCMV6-XL4-BIG2 WT and inactive mutants AKAP-A, -B, -C have beendescribed (37).BIG1 AKAP domain mutation was introduced using Quik-

Change II XL site-directed mutagenesis kit (Agilent Technologies)with HA-BIG1(V583W) primer 5′-ATATTTGAAAGATGGA-ATGATCTATCAAAA-3′. β-catenin S675 mutations were simi-larly introduced using QuikChange II site-directed mutagenesis kit(Agilent Technologies) with primers 5′-TACAAGAAACGGCT-TGACGTTGAGCTGACCAGC-3′ (S675D) and 5′-TACAAG-AAACGGCTTGCAGTTGAGCTGACCAGC-3′ (S675A).

Cell Culture and Transfection. HeLa cells used in all experiments,grown in DMEM (Invitrogen) with 10% (vol/vol) FBS (Invitrogen)in a humidified 5% (vol/vol) CO2 atmosphere at 37 °C, weretransfected with 100 nM BIG1- or BIG2-specific siRNA. Sensesequences were GUCCAAAUGUCCUCGCAUA (BIG1) andCCAAAAGAUUGACCGAUUA (BIG2), with DharmaconsiCONTROL nontargeting siRNA as negative control. DharmaFECT1transfection reagent was used according to manufacturer’s in-structions with a 72-h incubation. HilyMax DNA transfectionreagent (Dojindo Molecular Technologies) was used 24 h beforecell analysis for transient protein expression according to themanufacturer’s protocol.

Western Blotting and Immunoprecipitation.To quantify protein (BCAkit, Pierce), collected cells were broken in radio-immunoprecipi-tation assay (RIPA) buffer (50 mM Tris·HCl, pH 7.4, 150 mMNaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS,and EDTA-free protease inhibitor mixture). Samples of proteins(15 μg) were separated by SDS/PAGE, identified by Westernblotting, and quantified by densitometry.To cross-link interacting proteins before IP, cells were incubated

(30 min, room temperature) with 2 mM dithiobis-succinimidyl-

propionate (Pierce), a thiol-cleavable cross-linker dissolved in dryDMSO, which was quenched with 20 mM Tris buffer, pH 7.5,before cells were mechanically broken in ice-cold Nonidet P-40buffer (50 mM Tris·HCl, pH 7.4, 50 mM NaCl, 1% Nonidet P-40and EDTA-free protease inhibitor mixture). Samples of proteins(100 μg) were incubated overnight (4 °C with mixing) with 1 μg ofspecific antibodies or control IgG, and 3 h more after adding15 μL of Dynabeads Protein G (Invitrogen). Beads were washedfive times with Nonidet P-40 buffer before proteins were eluted,separated by SDS/PAGE in 4–12% NuPAGE gel (Invitrogen),transferred to nitrocellulose membranes (Invitrogen) that wereblocked with 5% (wt/vol) nonfat milk (Bio-Rad), reacted withprimary and secondary (horseradish peroxidase-coupled) anti-bodies, developed using Super Signal Chemiluminescent substrate(Pierce), and exposed in the LAS-4000 system (Fujifilm). In mostexperiments, samples of input (5%, before IP) and of proteinsfrom IP (50%) were analyzed.

Yeast Two-HybridAnalyses.Yeast two-hybrid analyses were performedin Saccharomyces cerevisiae strain AH109 (Clontech) according tothe manufacturer’s instructions. Briefly, after transformation withpGADT7 and pGBKT7 fusion constructs plus carrier DNA usinglithium acetate, yeast was grown on nonselective plates (lackingleucine and tryptophan) and colonies were transferred to selectiveplates (lacking histidine and/or adenine).

In Vitro Protein Synthesis and Binding Assays. TNT coupled wheatgerm extract system (Promega) was used for transcription accordingto the manufacturer’ s recommendations. Reactions containingtemplate DNA 1 μg, wheat germ extract 25 μL, reaction buffer2 μL, RNA polymerase (SP6 or T7) 1 μL, 1 μL of 1 mM amino acidmixture minus methionine, 1 mM amino acid mixture minus leu-cine 1 μL, and nuclease-free water to a total volume of 50 μL, wereincubated (30 °C, 2 h). For in vitro binding reactions, 20-μL sam-ples of protein products were incubated (4 °C, 4 h) in Nonidet P-40buffer before IP and analysis by Western blotting.

Subcellular Fractionation.Fractionation of postnuclear supernatant(PNS) by density-gradient centrifugation has been described (31).Briefly, cells (90% confluent, 10-cm plates) were washed withPBS, harvested by scraping in ice-cold homogenization buffer(0.25 M sucrose, 10 mM Tris-Cl, pH 7.4, 1 mM magnesium ace-tate), incubated on ice (15 min), and broken by passage ∼15 timesthrough a 30-gauge needle to ∼90% complete breakage. Ho-mogenates were centrifuged (800 × g, 6 min, 4 °C) to removenuclei and unbroken cells; samples (0.6 mL) of PNS were trans-ferred to step gradients containing 2%, 6%, 10%, 14%, 18%,22%, and 26% (wt/vol) iodixanol (0.6 mL each, total volume 4.2mL). After centrifugation (100,000 × g, 4 °C, 3 h), 15 fractionswere collected from the top and 15-μL samples of each weresubjected to SDS/PAGE for analysis by Western blotting anddensitometric quantification.

Confocal Immunofluorescence Microscopy. Cells (7 × 104 per well)were grown on Lab-Tek four-well chamber slides (Nunc) 24 hbefore fixation [4% (wt/vol) paraformaldehyde in PBS, 15 min,25 °C] and permeabilization (0.01% Triton X-100 and 0.05%SDS in PBS, 5 min). After 30 min in blocking buffer (0.1% sa-ponin, 0.2% BSA in PBS), cells were incubated with primaryantibodies in the same buffer (1 h), washed with PBS, incubatedwith Alexa Fluor-conjugated secondary antibodies in blockingbuffer (1 h), and mounted in VECTASHIELD mounting me-dium with DAPI (Vector Laboratories). Images were acquired

Li et al. www.pnas.org/cgi/content/short/1601918113 1 of 8

using LSM 510 META laser confocal microscope (Carl Zeiss)with 40×/1.3 N.A. oil objective lens and 488- or 543-nm lasers.Pinholes were set to scan ∼1-μm layers (resolution 512 × 512pixels). Projection view and optical sections were processeddigitally using CLSM5 Zeiss Browse Image software. Figureswere assembled and labeled using Adobe Photoshop (AdobeSystems).

RNA Extraction, Reverse Transcription, and PCR. Total RNA andfirst-strand cDNA were prepared using RNeasy Plus mini kit andQuantiTect reverse transcription kit (Qiagen) according to man-ufacturer’s instructions. Semiquantitative reverse transcription-PCR was performed to estimate β-catenin/TCF target gene (c-myc,and cyclin D1) mRNA levels. Primer sequences (forward and re-verse) were: β-actin, 5′-CTGGGACGACATGGAGAAAA-3′ and5′-AAGGAAGGCTGGAAGAGTGC-3′; c-myc, 5′-ATTTCTAC-TGCGACGAGGAGG-3′ and 5′-TGCTGTCGTTGAGAGGGT-AGG-3′; and cyclin D1, 5′-CTGGAGCCCGTGAAAAAGAGC-3′ and 5′-CTGGAGAGGAAGCGTGTGAGG-3′. For PCR (totalvolume 50 μL), we used ∼1 μg of cDNA, 5 pmol of each primer,200 μM of each dNTP, and 1 unit of Taq polymerase (TaKaRa).After denaturation (95 °C, 3 min), 25 PCR cycles of 95 °C/40 s,53 °C/40 s, and 72 °C/40 s ended with 10 min at 72 °C. PCRproducts were subjected to electrophoresis in 2% agarose gel inTBE buffer (89 mM Tris-base, pH 7.6, 89 mM boric acid, 2 mM

EDTA), before staining with ethidium bromide and photographyon a UV light box.

Luciferase Assays of β-Catenin Transcriptional Activity. Cells growingin 12-well plates were transiently transfected with TOP-Flash or

FOP-Flash reporter plasmid (1 μg), containing eight copies ofoptimal or mutant TCF/LEF consensus-binding sites upstreamof firefly luciferase, together with Renilla luciferase reporterplasmid (pRL-TK, 0.1 μg), using dual luciferase assay kit (Prom-ega), according to the manufacturer’s instructions. After 24 h, cellswere collected and lysed in passive lysis buffer (Promega). Fireflyluciferase activity (TCF-responsive reporter TOP-Flash) of eachsample was normalized for transfection efficiency to its Renillaluciferase activity driven by TK promoter.

Cell-Surface Protein Isolation. To isolate biotinylated cell-surfaceproteins, Pierce Cell Surface Protein Isolation Kit (89881, ThermoScientific) was used according to the manufacturer’s protocol. Forlabeling surface proteins, cells on 100-mm plates were washedthree times with ice-cold PBS and incubated (30 min, 4 °C) in10 mL of PBS containing 0.24 mg/mL EZ-link sulfo-NHS-SS-biotin before reaction-quenching solution (Thermo Scientific)was added. Cells were then washed twice with TBS, collected byscraping gently, and homogenized in 400 μL of lysis buffer(Thermo Scientific). Cell lysates (300 μg of protein in 300 μL)were mixed end over end (1 h, room temperature) with 300 μL ofimmobilized NeutrAvidin agarose beads (Thermo Scientific),which were then washed and biotinylated proteins eluted in300 μL of SDS sample buffer containing 50 mM DTT. Samples(20 μL) were subjected to SDS/PAGE and proteins analyzed byimmunoblotting.

Statistical Analysis.Data are means ± SEM of values from at leastthree experiments with significance defined as P < 0.05 usingStudent’s t test.

Fig. S1. Density gradient distribution of endogenous HeLa cell proteins: BIG1, BIG2, and β-catenin. (A) Proteins in postnuclear supernatants of cells separatedin iodixanol gradients as described in Materials and Methods. Lower gradient numbers correspond to lower and higher to greater densities. Amounts ofprotein are percentage in each fraction of the total amount of that protein recovered. (B) Cells were fixed and reacted with rabbit antibodies against BIG1 orBIG2 and mouse anti–β-catenin antibodies. (Scale bar, 10 μm.) (C) Quantification of Fig. 2A. β-Catenin in cells was quantified as the ratio of mean β-cateninstaining intensity of Golgi to mean β-catenin staining intensity of cytosol (Golgi/cytosol). At least 20 cells were analyzed in each of three experiments. ***P <0.005; *P < 0.05 vs. NT siRNA.

Li et al. www.pnas.org/cgi/content/short/1601918113 2 of 8

Fig. S2. Effects of BIG1 and/or BIG2 depletion on amounts of N-cadherin and Y654-phosphorylated (pY654) β-catenin. (A) After a 72-h incubation with siRNA,nontarget (NT) or specific for BIG1 or BIG2, or both, cells were fixed, reacted with antibodies against N-cadherin and GM130, and prepared for confocalimmunofluorescence microscopy. (Scale bar, 10 μm.) (B) Samples (20 μg) of total lysate proteins were separated in 4–12% NuPAGE Bis·Tris gels, reacted withindicated antibodies, and quantified by densitometry; values were normalized to that of control (NT siRNA) cells = 1.0. Data are means ± SEM (n = 3), *P < 0.05vs. NT siRNA. (C) Cells were incubated 72 h with NT or specific BIG1 and/or BIG2 siRNA before analyses of indicated proteins by Western blotting with anti-bodies against pY654 β-catenin (pY654–β-cat), densitometric quantification, and statistical analysis of data from three experiments as in B.

Li et al. www.pnas.org/cgi/content/short/1601918113 3 of 8

Fig. S3. Effects of overexpressed GEF-inactive BIG1 or BIG2 mutants, but not BIG1 AKAP mutant, on cellular distribution of β-catenin. After a 24-h transfectionwith cDNA encoding HA-tagged BIG1 WT or mutants E793K, or AKAP (A), or GFP-tagged BIG2 WT or mutant E738K (B), cells were fixed, reacted with anti-bodies against β-catenin or GM130 (cis-Golgi marker), and HA or GFP, and prepared for confocal immunofluorescence microscopy. Perinuclear accumulation ofβ-catenin in cells expressing BIG1 (E793K) or BIG2 (E738K), but not WT BIG1 or BIG2 or BIG1 AKAP mutant, is consistent with blockage of Arf activation atmembranes in a β-catenin translocation pathway. (Scale bar, 10 μm.)

Li et al. www.pnas.org/cgi/content/short/1601918113 4 of 8

Fig. S4. Effects of BIG1 and/or BIG2 depletion on levels of specifically serine-phosphorylated and total β-catenin or Akt. (A and B) Cells were incubated 72 hwith vehicle alone (mock) or with NT or specific BIG1 and/or BIG2 siRNA before analyses of indicated proteins by Western blotting and densitometric quan-tification. *P < 0.05; **P < 0.01 vs. NT siRNA.

Fig. S5. Overexpression of BIG1-F, BIG1-N, but not BIG1 (E793K) mutant, or BIG1-S, or -C, reversed effects of BIG1 or BIG2 depletion on levels of pS675β-catenin. (A) After a 72-h depletion of BIG1, cells were incubated 24 h with 4 μg of empty vector (EV) or 4 or 2 μg of HA-BIG1 E793K mutant before analysis ofproteins. *P < 0.01 vs. NT siRNA. (B) Western blots of proteins from cells after 72 h with BIG1 siRNA followed by 24 h with plasmids (4 μg) for expression of,respectively, BIG1-full length (F), -N terminus (N), -Sec7 domain (S), or -C terminus. *P < 0.05 vs. NT siRNA; **P < 0.05 vs. BIG1 siRNA.

Li et al. www.pnas.org/cgi/content/short/1601918113 5 of 8

Fig. S6. Effects of BIG1 and/or BIG2 depletion on cellular localization of active β-catenin. After a 72-h incubation with vehicle (mock) or siRNA, NT, or specific forBIG1 or BIG2, or both, HeLa cells were fixed, reacted with antibodies against β-catenin (total) or active β-catenin (without phosphorylation of Ser37 and Thr41), andprepared for confocal immunofluorescence microscopy. (Scale bar, 10 μm.) To quantify active β-catenin in nuclei of HeLa cells, nuclear perimeter was demarcatedand fluorescence intensity quantified in pixels using ImageJ software. In each group, >50 transfected cells were counted. ***P < 0.005 vs. NT siRNA.

Li et al. www.pnas.org/cgi/content/short/1601918113 6 of 8

Fig. S7. Effects of BIG1 and/or BIG2 depletion on Wnt3a-induced transcriptional activity and amounts of cell-surface receptor proteins. (A) After a 48-hdepletion of BIG1 and/or BIG2, cells were incubated for 24 h with TOP-Flash or FOP-Flash, together with pRL-TK reporter plasmids followed by a 1-h incubationwith 100 ng/mL Wnt3a, before TCF/LEF reporter activity was measured as described inMaterials and Methods. *P < 0.01 vs. NT siRNA, **P < 0.05 vs. NT siRNA +Wnt3a. (B) Cells were incubated (72 h) with vehicle (mock) or siRNA, NT, or specific for BIG1 or BIG2 or both. Samples (20 μg) of total cell lysate or biotinylatedcell-surface proteins from replicate samples were separated in 4–12% Bolt Bis·Tris Plus gels (Invitrogen), followed by reaction with indicated antibodies. (C andD) Amounts of LRP6, Frizzled 6, and integrin β1 in total cell lysate (C) or at cell surface (D) from three experiments like that in B were quantified by densi-tometry and values expressed relative to that of control (mock) cells = 1.0. Data are means ± SEM, *P < 0.05, **P < 0.01 vs. NT siRNA.

Li et al. www.pnas.org/cgi/content/short/1601918113 7 of 8

Fig. S8. Effects of BIG1 and/or BIG2 depletion on β-catenin–induced transcriptional activity and BIG2 AKAP-C mutant on phosphorylation of β-catenin S675. Aftera 48-h depletion of BIG1 or BIG2, cells were transfected (24 h) without or with 4 μg of EV or β-catenin (S675D) phosphomimetic mutant before measurement of TCF/LEF reporter activity (A). *P < 0.05 vs. NT siRNA, **P < 0.05 vs. BIG1 siRNA + EV or BIG2 siRNA + EV. Expression of β-catenin mutant (S675A) that cannot bephosphorylated was, however, ineffective (B). *P < 0.05 vs. EV. (C and D) After a 48-h depletion of BIG1 or BIG2, cells were transfected (24 h) with EV or appropriateWT BIG1 or BIG2 followed by 24-h incubation with TOP-Flash or FOP-Flash together with pRL-TK reporter plasmids. During a final 1-h incubation, 10 μM H-89protein kinase inhibitor or DMSO (vehicle) was present, as indicated, before measurement of TCF/LEF reporter activity as in A. *P < 0.05 vs. NT siRNA, **P < 0.05 vs.BIG1 siRNA + EV or BIG2 siRNA + EV. (E and F) After a 48-h depletion of BIG1 or BIG2, cells were transfected (24 h) with 4 μg of EV, BIG1 AKAP mutant (E), orindicated BIG2 AKAP mutant (F), and TCF/LEF reporter activity was measured as in A. *P < 0.05 vs. NT siRNA, **P < 0.05 vs. BIG1 siRNA + EV or BIG2 siRNA + EV.

Li et al. www.pnas.org/cgi/content/short/1601918113 8 of 8