Post on 04-Aug-2020
Supplement figure legendsFigure S1 Expression of IFN-β mRNA by RIG-I and Riplet expression
Riplet, RIG-I, dRIG-I or TICAM-1 encoding plasmids were transfected into HEK293cells, and 24 hours after transfection, total RNA was extracted and endogenous IFN-β
mRNA was examined by RT-PCR.
Figure S2 Cytoplasmic localization of Riplet and RIG-I
HA-tagged and FLAG-tagged RIG-I were expressed in HeLa cells, and their
intracellular localizations were observed by confocal microscope with anti-FLAG or
anti-HA antibodies. Riplet and RIG-I are localized in the cytoplasm but not nucleus.
Figure S3 Knockdown of Riplet
HA-tagged Riplet (100 ng) and siRNA ( 10 pmol) were transfected into HEK293 cells
in 24-well plate, and 48 hours after transfection, cell lysate was prepared and analyzed
by SDS-PAGE. HA-tagged Riplet was abolished by siRNA for Riplet (Riplet siRNA or
Riplet si-1) but not control si or control si-1 (upper panel). Total protein was stained by
CBB (lower panel).
Figure S4
(A) HEK293FT cells were transfected with RIG-I dRD (100 ng), Riplet (100 ng) and
HA-tagged ubiquitin (300 ng) in 6-well plate, and after 24 hours, immunoprecipitation
was carried out with anti-FLAG antibody and the precipitates were analyzed by western
blotting.
(B) HEK293FT cells were transfected with RIG-IC (100 ng), Riplet (100 ng) and HA-
tagged K63R or K48R mutant ubiquitin, after 24 hours, immunoprecipiation was
carried out with anti-FLAG antibody and then the precipitates were analyzed by western
blotting.
(C) HEK393FT cells were transfected with FLAG-tagged RIG-IC, HA-tagged Riplet-
DN, HA-Ub and/or HA-tagged Riplet and then after 24 hours, immunoprecipitation was
carried out with anti-FLAG antibody. The immunoprecipitates was analyzed by western
blotting.
Figure S5
HEK293FT cells were transfected with dRIG-I, RIG-I RD, HA-tagged ubiquitin and/or
Riplet. After 24 hours, the cell lysate were prepared and immunoprecipitation was
carried out with anti-FLAG antibody. The immunoprecipitates were analyzed by
western blotting with anti-HA or FLAG antibodies.
Figure S6
(A) Schematic representations of RIG-I 3KA and 5KA mutants. The magnification of C-
terminal region of RIG-I mutants are shown in the lower panel. The lysine residues at
888, 907 and 909 were replaced with alanine residues in RIG-I 3KA mutant. RIG-I 5KA
mutant harbors five amino acids substitutions of alanine residues for the lysine residues,
849, 851, 888, 907 and 909.
(B) The amino acids sequence of the RIG-I C-terminal region of RIG-I. The mutated
lysine residues, 849, 851, 888, 907 and 909, are bolded and underlined.
SupplementsMaterials and Methods
Confocal microscope
HeLa cells were plated onto coverslips in a 24-well plate. After cells were
adhered onto the coverslips, cells were transfected with plasmid encoding FLAG-tagged
RIG-I and HA-tagged Riplet. After 24 hours incubation, cells were washed twice in
PBS and fixed with PBS containing 3% formaldehyde for 30 minutes at room
temperature. After the fixation, cells were washed with PBS and permialized with 0.2 %
Triton X-100 in PBS for 15 minutes. The cells were washed with PBS, and coated with
1 % BSA in PBS for 10 minutes at room temperature. Primary antibodies were diluted
500 times with 1 % BSA, and the cells were incubated with the diluted primary
antibody for one hour. After three times washing with 1 % BSA in PBS, the cells were
incubated with secondary antibodies, Alexa Fluor 488 rabbit and Alexa Fluor 594
mouse antibody, for 30 minutes at room temperature. The coverslips were washed three
times with 1 % BSA in PBS, and then mounted onto slideglasses using 2.3 % DABCO
in PBS. Imaging of the cells were carried out using Olympus Fluoview laser scanning
confocal microscopy.
RT-PCR to detect IFN-β mRNA
HEK293 cells were plated onto 24-well plate. Cells were transfected with the
pEF-BOS expression vector encoding dRIG-I, full-length RIG-I, Riplet and/or TICAM-
1 by FuGENE HD reagent. 24 hours after transfection, total RNA was extracted using
TRIZOL reagent. cDNA was generated by M-MLV-reverse transcriptase (Promega)
with random 9mer, and then subjected to PCR using Ex-Taq (TAKARA). The DNA
sequences of primers are IFN-beta forward: ATT GCC TCA AGG ACA GGA TG, IFN-
beta reverse: CTA TGG TCC AGG CAC AGT GA, GAPDH forward: CAC AGT CCA
TGC CAT CAC TG and GAPDH reverse: TAC TCC TTG GAG GCC ATG TG.
Preparation of HCV poly-U/UC RNA
The HCV genotype 1b poly-U/UC RNA (from 9421 to 9480, Accession number:
EU867431) was synthesized by T7 RNA polymerase in vitro. The template dsDNA
sequences were; Forward: TAA TAC GAC TCA CTA TAG GGT TCC CTT TTT TTT
TTT CTT TTT TTT TTT TTT TTT TTT TTT TTT TTT CTC CTT TTT TTT TC,
Reverse: GAA AAA AAA AGG AGA AAA AAA AAA AAA AAA AAA AAA AAA
AAA AGA AAA AAA AAA AGG GAA CCC TAT AGT GAG TCG TAT TA. The
synthesized RNA was purified by TRIZOL reagent (Invitrogen).
1
2
Figure S3
Rip
let s
iRN
A
Con
trol s
iRN
A
Rip
let s
i-1
Con
trol s
i-1
(A)
UbRIG-IdRD
*
anti-HA anti-FLAG(Ub) RIG-I dRD
RIG-I dRD + + + +Riplet + - + -HA-Ub + + + +
(B)
IP:FLAG (RIG-IC)WB:HA (HA-Ub)
IP:FLAG (RIG-IC)WB:FLAG (RIG-IC)
Ub
RIG-IC
RIG-IC + + -Riplet + + - HA-Ub K48R K63R WT
(C)
Figure S4
WB: FLAG
WB: HA
WB: FLAGWB: HA
WB: HA
<WCE>
<IP: FLAG>
Riplet + - + - + -dRIG-I + + - - - -RIG-I RD - - + + - -HA-Ub + + + + + +
Ub-RIG-I
Riplet
dRIG-IRIG-I RD
RipletdRIG-IRIG-I RD
Fig. S5
888
XX X
XX X
907909
735 925
851
3KA
(A)
(B)
5KA
888 907 909849
1
XX
735 925
RIG-I 3KA
RIG-I 5KA