Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc...
Transcript of Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc...
Regulation of the linear ubiquitination of STAT1 controls antiviral interferon signaling Zuo et al.
Supplementary Information
Supplementary Figure 1
a
b c
d
e
Flag-HOIP + + + + + +Myc-STAT1 Δ24
1-310
Δ431-
490
Δ1-13
5Δ1-3
24
IP IgG Myc
Myc
Flag
Flag
Tubulin
43
557295IP
Inpu
t
Flag-HOIP + + + + + + +Myc-STAT1 Δ33
6-413
Δ491-5
76Δ57
7-657
Δ658-7
03Δ70
4-750
IP IgG Myc
72
95
Flag
Myc
IP
Flag
TubulinInpu
t
f g
750N-ter CC DB Linker SH2 TAD WT
Δ1-135Δ1-324
Δ241-310Δ336-413Δ431-490Δ491-576
Δ658-703
1 135 317 488 576 703
Δ704-750
WT-STAT1
Δ577-657
WT
WT
WT
WT
shHOIP - #1 #2 -Flag-HOIP + + + +
Flag
Tubulin
IB: IB:
HA-K0 + + + Myc-STAT1 + + +
shHOIP IP IgG Myc
HA
Myc
HOIP
Tubulin
130
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IP
HA
Inpu
t
- - +
IB:Flag-LUBAC - - +
IP IgG STAT1
ub
STAT1
Flag-HOIP
Flag-HOIL-1LFlag-Sharpin
Tubulin
IPIn
put
130
180IB:Myc-STAT1 + + +
shHOIP - -IP IgG Myc
Line
ar u
b
Myc
IP
HOIP
Tubuin
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180
Inpu
t
IB:
+
IP IgG STAT1HOIP
STAT1
IP
293T
HOIP
STAT1
IP IgG STAT1
IP
2fTGH
HOIP
TubulinInpu
t
HOIP
TubulinInpu
t
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95130
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200 400 600 800 1000m/z0
100200300400500600700800
Inte
nsity
[cou
nts]
(103
)
1200
Supplementary Figure 1
STAT1 has linear ubiquitination mediated by HOIP.(a) Mass spectrometry analysis of the HOIP protein interacting with STAT1. (b) Immunoprecipitation (IP) and immunoblotting (IB) analysis of the interaction between Flag-HOIP and Myc-STAT1(WT) or its different deletion mutants (Δ1-135, Δ1-324, Δ241-310, Δ336-413, Δ431-490, Δ491-576, Δ577-657, Δ658-703, Δ704-750) in HEK293T cells. (c) Immunoprecipitation analysis of the interaction between endogenous HOIP and STAT1 in 2fTGH and HEK293T cells. (d) Western blot analysis of Flag-HOIP protein levels in HEK293T cells cotransfected with shHOIP (#1, #2) and Flag-HOIP. (e) Immunoprecipitation analysis of ubiquitination of STAT1 in HEK293T cells cotransfected with Myc-STAT1, shHOIP and HA-Ub-K0 (HA-K0, all lysines on Ub are mutated to arginine). (f) Immunoprecipitation analysis of linear ubiquitination of STAT1 in HEK293T ce l l s co t r ans fec t ed wi th shHOIP and Myc-STAT1 . (g ) Immunoprecipitation analysis of ubiquitination of STAT1 in HEK293T cells transfected with or without Flag-LUBAC. Data are representative of three independent experiments (b-g).
Supplementary Figure 2
a cS
tat1
mR
NA
1.5
1.0
0.5
0.0
1.5
1.0
0.5
0.0
Sta
t2 m
RN
A
d e
i
g
h j
k l
shHOIP(#1)shCON
shHOIP(#2)
shHOIP(#1)shCON
shHOIP(#2)
shCON shHOIPIFNα - + - +
IFIT1
HOIP
Actin
PKR
IFNɑ - + - + - + - + 0.0
0.5
1.0
1.5
HS
V R
NA
** **
****
Icp27 Ul46
shHOIP shCON shCON shHOIP
0
1
2
3
4
Mx1
mR
NA **
*
shHOIP(#1)shCON
shHOIP(#2)0.0
0.5
1.0
1.5
Vira
l RN
A
*
virus invasion,1 hrVSV SeV HSV
* **
shCONshHOIP
shCONshHOIP
shCONshHOIP
0.0
0.5
1.0
1.5
Vira
l RN
A
* *** **VSV SeV HSV
virus invasion, 1 hr
LUBACCON
LUBACCON
LUBACCON
N.S N.S
shHOIP(#1)
shHOIP(#2)
shHOIP(#1)
shHOIP(#2)0.0
0.5
1.0
1.5
VS
V R
NA
N.S
0.0
0.5
1.0
1.5
SeV
RN
A
N.SU3A (Stat1-/-)U3A (Stat1-/-)
shHOIP - #1 #2
HOIPTubulin
shCONshCON
U3A (Stat1-/-)
-IFNα +IFNα -IFNα +IFNαCON LUBAC
VSV-GFP VSV-GFP VSV-GFP VSV-GFP
IFNα (min) 0 30 60 0 30 60
HOIP
shCON shHOIP
Tubulin
pY-STAT1
STAT1
shHOIP(#1)
0
2
4
6
Isg5
4 m
RN
A
***
IFNα - + + +
shCONshCON
shHOIP(#2)
Flag-LUBAC - - IFNβ - - + +
+ ++
pY-STAT1
STAT1
Flag-HOIP
Flag-HOIL-1L
Flag-Sharpin
Tubulin
b
05
10
15
20
Ifit1
mR
NA
IFNα - + +
CONCON
LUBAC
***f
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Supplementary Figure 2
Linear ubiquitination sustains STAT1 signaling homeostasis. (a) RT-qPCR analysis of Stat1 and Stat2 mRNA in HEK293T cells transfected with control shRNAs (shCON) or shHOIP (#1, #2). (b) Western blot analysis of pY701-STAT1 (pY-STAT1) levels in HEK293T cells transfected with increasing amounts of Flag-LUBAC, and then treated with IFNβ (1,000 IU/ml) for 30 min. (C) Western blot analysis of pY-STAT1 in HeLa cells transfected with shHOIP and then treated with IFNα (1,000 IU/ml) for the indicated times. (d) RT-qPCR analysis of Isg54 mRNA in HEK293T cells transfected with either shCON or shHOIP (#1, #2) and then stimulated with IFNα (1,000 IU/ml) for 4 hrs. (e) Western blot analysis of the representative ISGs proteins (IFIT1 and PKR) in HEK293T cells transfected with shHOIP and then treated with IFNα (500 IU/ml) for 24 hrs. (f) RT-qPCR analysis of Ifit1 mRNA in HEK293T cells transfected with vectors (CON) or LUBAC-expressing constructs and then stimulated with IFNα (1,000 IU/ml) for 4 hrs. (g) RT-qPCR analysis of HSV viral Icp27 and Ul46 RNA in 2fTGH cells transfected with shHOIP and then treated with IFNα (60 IU/ml) for 20 hrs, followed by infection with HSV (MOI=1.0) for 24 hrs. (h) Fluorescence microscopy of VSV-GFP in A549 cells transfected with empty vectors (CON) or Flag-LUBAC and then infected by VSV-GFP (MOI=0.1) for 24 hrs. Scale bar: 100 µm. (i) RT-qPCR analysis of a representative ISG (Mx1) mRNA in HEK293T cells transfected with shHOIP (#1, #2). (j, k) RT-qPCR analysis of viral RNA in 2fTGH cells transfected with either shHOIP (j) or Flag-LUBAC (k) and then infected by VSV (MOI=0.1), SeV (MOI=1.0) or HSV (MOI=1.0) for 1 hr to observe virus invasion. (l) RT-qPCR analysis of viral RNA in U3A cells transfected with shHOIP (#1, #2) and then infected by VSV (MOI=0.1) or SeV (MOI=1.0) for 1 hr. HOIP knockdown was analyzed by Western blot. N.S, not significant (p>0.05) and *p<0.05, **p<0.01, ***p<0.001 (two-tailed unpaired Student’s t-test). Data are shown as mean and s.d. of three biological replicates (a, d, f, g, i-l), or are representative of three independent experiments (b, c, e, h).
Supplementary Figure 3
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d e f
shHOIP - #1 #2Flag-IFNAR1 - + + +
Tubulin
Flag
HOIP
- shHOIP - #1 #2HA-IFNAR2 - + + +
Tubulin
HOIP
HA
-HA-IFNAR2 - + + +
Flag-LUBAC -
HA
Flag-HOIP
Flag-HOIL-1LFlag-Sharpin
Tubulin
- + ++
HA-K0 + + + +Myc-STAT1
IP IgG Myc
HA
Myc
K511RK652R
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HA
Tubulin
Inpu
tIP
WTWT
IB:
shHOIP - #1 #2
p-STAT2
Tubulin
IFNα - + + +
HOIP
-
STAT2
Flag-STAT1 DM
pY-STAT1
Tubulin
IFNβ (min) 0 30 60 0 30 60
Flag
U3A (Stat1-/-)WT
0.00.5
1.0
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2.5 **Ifit1 Isg15 Isg54
ISG
s m
RN
A
Myc-STAT1
** ***
DMWT DMWT WT DM
i
h
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1.0
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VS
V R
NA
IFNɑ - + - + - +Flag-STAT1 - - DM DMWT
**
****
WT
U3A (Stat1-/-)
ND CCD DBD LD SH2D TAD012345
136 317 488 576 683 750
K193
K240 K2
96 K410
K511
K592
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Log2
(num
ber o
f ref
eren
ces)
STAT1 gene sites
g
Flag-LUBAC - - IFNγ - - + +
+ ++
pY-STAT1
Flag-HOIL-1LFlag-Sharpin
Tubulin
STAT1
Flag-HOIP
IFNγ (min) 0 30 60 0 30 60
Myc-STAT1 DMU3A (Stat1-/-)
WT
pY-STAT1
Tubulin
Myc
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20N.S
Ifit1
mR
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IFNγ - + + +
shCON
shHOIP(#1)shCON
shHOIP(#2)
N.S
02
4
6
8
10**
N.S
IFNγ - + - +
Myc-STAT1WT DM
Ifit1
mR
NA
U3A (Stat1-/-)k
jl m
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Supplementary Figure 3
LUBAC induces STAT1 linear ubiquitination at Lys511/652.(a) Western blot analysis of Flag-IFNAR1 levels in HEK293T cells cotransfected with Flag-IFNAR1, together with shCON (-) or shHOIP (#1, #2). (b,c) Western blot analysis of HA-IFNAR2 levels in HEK293T cells cotransfected with HA-IFNAR2 and either shHOIP (#1, #2) (b) or increasing amounts of Flag-LUBAC (c). (d) Western blot analysis of p-STAT2 in HEK293T cells transfected with shHOIP (#1, #2) and then treated with IFNα (1,000 IU/ml) for 30 min. (e) Putative ubiquitination sites of STAT1 obtained from the PhosphoSitePlus database. (f) Immunoprecipitation analysis of ubiquitination of Myc-STAT1 (WT, K511R, or K652R) in HeLa cells cotransfected with Myc-STAT1 (WT, K511R or K652R) and HA-K0 using a HA antibody. (g) Western blot analysis of pY-STAT1 in U3A cells transfected Flag-STAT1 (WT or DM) and then treated with IFNβ (1,000 IU/ml) as indicated. (h) RT-qPCR analysis of the representative ISGs (Ifit1, Isg15 and Isg54) mRNA in U3A cells transfected with Myc-STAT1 (WT or DM). (i) RT-qPCR analysis of VSV viral RNA in U3A cells transfected with Flag-STAT1 (WT or DM) and then treated with IFNɑ (60 IU/ml) for 20 hrs, followed by infection with VSV (MOI=0.1) for 24 hrs. (j) Western blot analysis of pY-STAT1 levels in HEK293T cells transfected with increasing amounts of Flag-LUBAC, and then treated with IFNγ (1,000 IU/ml) for 30 min. (k) Western blot analysis of pY-STAT1 in U3A cells transfected Myc-STAT1 (WT or DM) and then treated with IFNγ (1,000 IU/ml) as indicated. (l) RT-qPCR analysis of the representative ISG (Ifit1) mRNA in HEK293T cells transfected with shHOIP (#1, #2) and then stimulated with IFNγ (1,000 IU/ml) for 4 hrs. (m) RT-qPCR analysis of the representative ISG (Ifit1) mRNA in U3A cells transfected with Myc-STAT1 (WT or DM) and then treated with IFNγ (1,000 IU/ml) for 4 hrs. N.S, not significant (p>0.05), **p<0.01 and ***p<0.001 (two-tailed unpaired Student’s t-test). Data are shown as mean and s.d. of three biological replicates (h, i, l, m), or are representative of three independent experiments (a-d, f, g, j, k).
Supplementary Figure 4
a
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Flag-HOIP - + + +
IP Flag
Myc-STAT1 -
Myc
Flag
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TubulinIP
Inpu
t
IPIn
put
Flag-HOIP + + + +Flag
Myc
Flag
Tubulin
WTY701F
S727AMyc-STAT1 WT WT
Y701FS727A
IP IgG Myc
IB:IB:
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IP IgG Myc
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ar u
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IP
Myc
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Myc
TubulinInpu
t
pY-STAT1
pY-STAT1
HOIP
Lamb1
Tubulin
IFNα - + - +shHOIP
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lear
cyto
plas
m
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Flag-LUBAC - - IP IgG STAT1
IFNα + + +HA-IFNAR2 + +
HA
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IP
HA
Tubulin
Inpu
t
Flag-HOIP
Flag-HOIL-1L
Flag-Sharpin
IB:
Flag-LUBAC - - +IFNα + + +
IP IgG IFNAR2
IFNAR2
STAT1
Tubulin
Flag-HOIP
Flag-HOIL-1LFlag-SharpinIn
put
IP
IB:HA-IFNAR2 + + + +Myc-STAT1
HA
IP HA
Myc
Tubulin
MycIn
put
IP: H
A
- WT K511RK652R
IB:
IFNα + - + + + +
HA-IFNAR2 + + + + + +Flag-LUBAC
IP IgG HA
Myc
Flag-HOIP
Flag-HOIL-1L
Myc-STAT1 + + + + K511R K652R
HA
Flag-Sharpin
Myc
Tubulin
IP
Inpu
t
IB:
+ + +- --d
e f
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++
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Supplementary Figure 4
Linear ubiquitination blocks the binding of STAT1 to IFNAR2.(a) Western blot analysis of pY701-STAT1 (pY-STAT1) in the cytoplasm and nucleus of HEK293T cells transfected with shHOIP and then treated with IFNα (1,000 IU/ml) for 30 min. (b) Immunoprecipitation analysis of the interaction between Flag-HOIP and Myc-STAT1 (WT, Y701F or S727A) in HEK293T cells. (c) Immunoprecipitation analysis of linear ubiquitination of Myc-STAT1 (WT or Y701F) in HEK293T ce l l s t ransfec ted wi th Myc-STAT1 (WT or Y701F) . (d) Immunoprecipitation analysis of the interaction between HA-IFNAR2 and STAT1 in HEK293T cells transfected with Flag-LUBAC and then treated with IFNα (1,000 IU/ml) for 15 min. (e) Immunoprecipitation analysis of the interaction between endogenous IFNAR2 and STAT1 in HEK293T cel ls t reated as (d) . ( f ) Immunoprecipitation analysis of the interaction between HA-IFNAR2 and Myc-STAT1 (WT or its mutants) in HEK293T cells cotransfected with HA-IFNAR2 and Myc-STAT1 (WT, K511R or K652R). (g) Immunoprecipitation analysis of the interaction between HA-IFNAR2 and Myc-STAT1 (WT and its mutants) in HEK293T cells cotransfected with HA-IFNAR2, Flag-LUBAC and Myc-STAT1 (WT, K511R or K652R) and then treated with IFNα (1,000 IU/ml) for 15 min. Data are representative of three independent experiments.
Supplementary Figure 5
c
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0.5
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V R
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CON Flag-OTULIN
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IP IgG STAT1
HA
STAT1
Flag
Tubulin
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HA
Inpu
tIP
IB:
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IP IgG
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Inpu
t
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Flag-OTULIN + - +
STAT1
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IP IgG Flag
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Flag
TubulinInpu
t
IB:
IFNα - - - +Myc-STAT1 - + + +
Flag-OTULIN + - + +
Flag
Myc
Flag
Flag
Myc
Inpu
t
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IP
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aHA-K0 + + +
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HA
STAT1
IPIP IgG
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Tubulin
Inpu
t
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b293TSTAT1
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IFNα (min) 0 0 15 30HA-K0 + + + +
IP IgG STAT1
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STAT1IP IgG
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STAT1
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+ +
IFNα (min) 0 0 15 30
Line
ar u
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STAT1
IP IgG STAT1
IP
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IB:
d
jIFNα - + + +
pY-STAT1
Tubulin
OTULIN
shOTULIN - - #1 #2i
STAT1
STAT1
TubulinInpu
t
hIFNα (min) 0 0 10 20
IP IgG OTULIN
OTULIN
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IP
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STAT1
TubulinInpu
t
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Supplementary Figure 5
IFN-I removes STAT1 linear ubiquitination via OTULIN.(a) Immunoprecipitation analysis of ubiquitination of STAT1 in HEK293T (left) or HT1080 (right) cells transfected with HA-K0 and then treated with IFNα (1,000 IU/ml) for 30 min. (b) Immunoprecipitation analysis of ubiquitination of STAT1 in 2fTGH cells transfected with HA-K0 and then treated with IFNα (1,000 IU/ml) for the indicated times. (c) Immunoprecipitation analysis of linear ubiquitination of STAT1 in HEK293T cells treated with IFNα (1,000 IU/ml) as indicated. (d) Immunoprecipitation analysis of the interaction between Flag-OTULIN and STAT1 in HEK293T cells transfected with Flag-OTULIN. (e) Immunoprecipitation analysis of ubiquitination of STAT1 in HEK293T cells cotransfected with HA-K0 and Flag-OTULIN using a HA antibody. (f) Immunoprecipitation analysis of linear ubiquitination of STAT1 transfected with Flag-OTULIN using a linear ubiquitination antibody. (g) Immunoprecipitation analysis of the interaction between Flag-OTULIN and Myc-STAT1 in HEK293T cells transfected with Flag-OTULIN and Myc-STAT1 and then treated with IFNα (1,000 IU/ml) for 15 min. (h) Immunoprecipitation analysis of the interaction between endogenous OTULIN and STAT1 in HEK293T cells treated with IFNα (1,000 IU/ml) for the indicated times. (i) Western blot analysis of pY-STAT1 in HEK293T cells transfected with shOTULIN (#1, #2) and then treated with IFNα (1,000 IU/ml) for 30 min. (j) RT-qPCR analysis of VSV viral RNA in 2fTGH cells transfected with Flag-OTULIN and then stimulated with IFNα (60 IU/ml) for 20 hrs, followed by infection with VSV (MOI=0.1) for 24 hrs. **p<0.01 and ***p<0.001 (two-tailed unpaired Student’s t-test). Data are shown as mean and s.d. of three biological replicates (j), or are representative of three independent experiments (a-i).
Supplementary Figure 6
SeV (h) 0 3 6 12 24
HOIP
Tubulin
a b
c
d
VSV (h) 0 0 6 12IP IgG STAT1
HA
STAT1
HA-K0 + + + +
HA
Tubulin
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Inpu
tIP
IB:
SeV (h) 0 0 6 9
IP IgG STAT1
STAT1
HOIP
IP
IB:
HOIP
TubulinInpu
t
IP IgG STAT1
Line
ar u
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STAT1
IP
VSV (h) 0 0 6 12
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IB:
HOIP
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Inpu
t
Myc-STAT1 - WT WT WT YF YF YFSeV (h) 0 0 6 9 0 6 9
IP Myc
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ar u
bIB:
Myc
Tubulin
Myc
IP180
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Myc-STAT1 SeV - + - + - + - +
WT WT DM DM YF YF YF/DMYF/DM
IP Myc
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ar u
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IP: M
yc
pY-STAT1
pY-STAT1
Short exp.
Long exp.Myc
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t
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Rel
ativ
e Ifi
t1 m
RN
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Myc-STAT1 WT WT DM YF YF/DMSeV - + + + +
***
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Supplementary Figure 6
Viruses upregulate HOIP and STAT1 linear ubiquitination.(a) Western blot analysis of HOIP proteins in A549 cells infected with SeV (MOI=1.0) for the indicated times. (b) Immunoprecipitation analysis of the interaction between HOIP and STAT1 in HEK293T cells infected with SeV (MOI=1.0) for the indicated times. (c) Immunoprecipitation analysis of ubiquitination of STAT1 in HT1080 cells infected with HA-K0 and then infected with VSV (MOI=0.5) as indicated. (d) Immunoprecipitation analysis of linear ubiquitination of STAT1 in A549 cells infected with VSV (MOI=0.5) for the indicated times. (e) Immunoprecipitation analysis of l inear ubiquitination of STAT1 in HEK293T cells transfected with Myc-STAT1 (WT or YF) and then in fec ted wi th SeV (MOI=1 .0) a s ind ica ted . ( f ) Immunoprecipitation analysis of linear ubiquitination of STAT1 in U3A cells transfected with Myc-STAT1 (WT, DM, YF or YF/DM) and then infected with SeV (MOI=1.0) for 12 hrs. (g) RT-qPCR analysis of the representative ISG (Ifit1) mRNA in U3A cells transfected with Myc-STAT1 (WT, DM, YF or YF/DM) and then infected with SeV (MOI=1.0) for 12 hrs. (h) RT-qPCR analysis of SeV viral RNA in U3A cells transfected with Myc-STAT1 (WT, DM, YF or YF/DM) and then infected with SeV (MOI=1) for 12 hrs. N.S, not significant (p>0.05), **p<0.01 and ***p<0.001 (two-tailed unpaired Student’s t-test). Data are representative of three independent experiments (a-f), or are shown as mean and s.d. of three biological replicates (g, h).
Supplementary Figure 7
a
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Rbck1 +/+ +/+ +/-STAT1
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Mouse No. 1 2 1 2
Heart
HOIL-1L
Tubulin
Liver Spleen
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Rbck1+/- Rbck1+/+
Myc-STAT1 - WT WT DM WT DMmIFNβ - - + + + +
Rbck1+/+ Rbck1+/-
pY-STAT1
MycIP: M
yc
Myc
Tubulin
IB:In
put
HOIL-IL
012
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ifit1
mR
NA ******
mIFNβ - - + + + +
Rbck1+/+ Rbck1+/-
N.S
Myc-STAT1 - WT DM WT DMWT
WT K511/652R
eLentiviral packaging GFP-STAT1
GFP
-STA
T1
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Supplementary Figure 7
HOIL-1L deficiency enhances IFN-I antiviral activity.(a) Western blot analysis of HOIL-1L proteins in the heart, liver and spleen tissues from Rbck1+/+ or Rbck1+/- mice. (b) Immunoprecipitation analysis of linear ubiquitination of STAT1 in the heart tissues from Rbck1+/+ or Rbck1+/- mice. (c) Immunoprecipitation analysis of pY-STAT1 in Rbck1+/+ or Rbck1+/- MEF cells transfected with Myc-STAT1 (WT or DM) and then treated with mIFNβ (1,000 IU/ml) as indicated. (d) RT-qPCR analysis of the representative ISG (Ifit1) mRNA in Rbck1+/+ or Rbck1+/- MEF cells transfected with Myc-STAT1 (WT or DM) and then treated with mIFNβ (1,000 IU/ml) for 4 hrs. (e) The lentiviruses containing GFP-STAT1 (WT or K511/652R) were made in HEK293T cells. GFP signaling in cells was observed by the fluorescence microscopy. Scale bar: 100 µm. N.S, not significant (p>0.05) and ***p<0.001 (two-tailed unpaired Student’s t-test). Data are shown as mean and s.d. of three biological replicates (d), or are representative of three independent experiments (a-c, e).
Figure 1a
Figure 1bIB:Myc
95
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Figure 1d
IB:HOIP
IB:STAT1
IB:HOIP
IB:STAT1
IB:HOIP
IB:STAT1
130
95
95
Liver
130
95
95
Spleen
130
95
95
Lung
For Figure 1
IB:HOIP
IB:Tubulin
13095
55
IB:HOIP
IB:Tubulin
13095
55
IB:HOIP
IB:Tubulin
13095
55
Figure 1eIB:Linear ub
IB:STAT1
IB:HOIP
IB:Tubulin
95
130
180
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130
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IB:Linear ub
IB:STAT1
IB:Flag
IB:Tubulin
180
95
130
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43
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55
Figure 1f
180
130
IB:Linear ub 95
IB:STAT1
IB:HOIP
IB:Tubulin95130
55
Figu
re 1
g
130
Supplementary Figure 8: Original scans of immunblots.
For Figure 2
IB:STAT1
IB:Flag
IB:Tubulin
95
130
95
55
43
55
Figure 2a
95
55
13095
IB:STAT1
IB:HOIP
IB:Tubulin
Figure 2bIB:pY-STAT1
IB:Flag
IB:Tubulin
95
130
95
55
43
55
Figure 2cIB:pY-STAT1
IB:Tubulin
95
55
Figure 2d
IB:pY-STAT1
IB:Flag
IB:Tubulin
95
130
95
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43
55
Figure 2e
IB:VSV-G
IB:Flag
IB:Actin
72
130
95
55
43
43
Figure 2j
IB:pY-STAT1
IB:STAT1
IB:HOIP
IB:Tubulin
95
95
130
95
55
Figure 2l
IB:STAT1
95
IB:STAT1
IB:HOIP
95
13095
IB:STAT1
95
For Figure 3
IB:HA
IB:HA
IB:Flag
IB:Tubulin
180
130
180
72
13095
5543
55
Figure 3a
IB:HA
IB:JAK1
IB:HA
IB:Flag
IB:Tubulin
180
130
180
72
130
95
5543
55
IB:HA
IB:STAT1
IB:HA
IB:Flag
IB:Tubulin
130
180
95
180
72
13095
5543
55
IB:HA
IB:STAT2
IB:HA
IB:Flag
IB:Tubulin
180
130
180
72
13095
5543
55
IB:p-JAK1
IB:JAK1
IB:p-Tyk2
IB:Tyk2
IB:Flag
IB:Tubulin
130
130
130
130
130
5543
55
95
Figure 3b
IB:HA
IB:Myc
IB:HA
IB:Tubulin
180130
95
180
95
55
Figure 3c
IB:Linear ub
IB:Myc
180130
95
Figure 3eIB:Myc
IB:Flag
IB:Myc
IB:Tubulin
95
95
55
130
95
Figure 3f
130180 95
IB:Linear ub IB:Myc
IB:Flag1309555
43
55
IB:Tubulin
Figure 3g
IB:pY-STAT1
IB:Myc
IB:Tubulin
95
95
55Figure 3h
IB:Tyk2
95
IB:Myc
55IB:Tubulin
For Figure 4
IB:JAK1
IB:STAT1
IB:Flag
IB:Tubulin
130
95
130
95
55
43
55
Figure 4a
IB:JAK1
IB:STAT1
IB:HOIP
IB:Tubulin
130
95
130
95
55
Figure 4b
IB:JAK1
IB:STAT1
IB:HOIP
IB:Tubulin
130
130
95
95
55
IB:HA
IB:STAT1
IB:HOIP
IB:HA
IB:Tubulin
72
95
72
130
95
55
Figure 4c
IB:STAT1
IB:JAK1
95
130
IB:STAT1
IB:JAK1
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130
IB:STAT1
IB:JAK1
95
130
IB:STAT1
IB:IFNAR2
IB:HOIP
IB:Tubulin
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72
130
95
55
Figure 4d72
95
72
55
IB:HA
IB:Myc
IB:HA
IB:Tubulin
Figure 4e
72
95
IB:HA
IB:STAT1
Figure 4fIB:Flag
IB:HA
IB:Flag
95
72
95
Figure 4g
IB:STAT1
IB:IFNAR2
95
72
IB:pY-STAT1
IB:Flag
IB:HA
95
95
130
Figure 4h IB:Linear ub
IB:Flag
IB:Myc
130
180
95
72
Figure 4i
For Figure 5
95
IB:Linear ub
IB:pY-STAT1
IB:STAT1
95
130
180
Figure 5a
130
180
95
IB:Linear ub
IB:STAT1
Figure 5b
IB:Linear ub
IB:STAT1
IB:OTULIN
IB:Tubulin
55
43
95
130
180
Figure 5c
IB:Linear ub
IB:Myc
IB:OTULIN
IB:Tubulin
130
180
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43
55
Figure 5d
IB:STAT1
IB:Tubulin
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IB:Linear ub
IB:STAT1
IB:STAT1
IB:Tubulin
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95
43
IB:STAT1
IB:OTULIN
IB:STAT1
IB:Tubulin
95
55
Figure 5e
130
180
IB:Linear ub
IB:STAT1
IB:OTULIN
IB:Tubulin
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43
55
Figure 5f
180
IB:STAT1
IB:IFNAR2
IB:OTULIN
IB:Tubulin
55
43
72
95
Figure 5hIB:pY-STAT1
IB:OTULIN
IB:Tubulin
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43
95
Figure 5iIB:STAT1
IB:IFNAR2
IB:Flag
IB:Tubulin
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72
43
55
Figure 5g
IB:STAT1
95
For Figure 5
For Figure 6
IB:HOIP
IB:Tubulin
130
95
55
Figure 6dIB:HOIP
95
130
95
IB:STAT1
Figure 6eIB:Linear ub
IB:STAT1
130
180
95
Figure 6f
130180
IB:Linear ub
IB:STAT1
95
Figure 6g
IB:HOIP
IB:Tubulin95130
55
IB:HOIP
IB:Tubulin95130
55
IB:HOIP
95130
IB:Tubulin55
130
180
IB:Linear ub
IB:STAT1
95
Figure 6h
IB:pY-STAT1
IB:Tubulin
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55
Figure 6k
180130
IB:STAT1
IB:Linear ub
IB:HOIP
IB:Tubulin
55
13095
95
Figure 6j
IB:HOIP
95130
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IB:Tubulin
IB:HOIP
IB:STAT1
95
95130
IB:Linear ub
IB:Myc
IB:Myc
IB:Tubulin
130180
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95
55
Figure 6i
For Figure 7
130180
95
IB:Linear ub
IB:STAT1
130180
95
IB:Linear ub
IB:STAT1
130180
95
IB:Linear ub
IB:STAT1
Figure 7a
95
55
IB:pY-STAT1
IB:Tubulin
Figure 7b
55
IB:HOIL-1L
IB:STAT1
95
For Figure 8
IB:pY-STAT1
IB:GFP-STAT1
95
95
Figure 8b
For Supplementary Figure 1
IB:HOIP
IB:STAT1
95
130
95
IB:HOIP
IB:STAT1
95
95
130
Figure S1cIB:Flag
IB:Myc
IB:Flag
IB:Tubulin
130
95
55
95
130
95
55
IB:Flag
IB:Myc
IB:Flag
IB:Tubulin
55
130
95
95
72
130
95
Figure S1b
13095
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IB:Tubulin
IB:Flag
Figure S1d
IB:ub
IB:STAT1
IB:Flag
IB:Tubulin
55
5543
130
95
130
180
Figure S1g
IB:HA
IB:Myc
IB:HA
IB:HOIP
IB:Tubulin
55
130
95
95
180
95
130
180
Figure S1e
IB:Linear ub
IB:Myc
IB:HOIP
IB:Tubulin
55
130
95
95
130180
Figure S1f
IB:HOIP13095
IB:Tubulin
55
IB:HOIP
95130
55
IB:Tubulin
For Supplementary Figure 2
IB:pY-STAT1
IB:HOIP
IB:Tubulin
95
13095
95
Figure S2c
IB:IFIT1
IB:PKR
IB:HOIP
IB:Actin
55
72
130
95
43
Figure S2e
IB:HOIP
IB:Tubulin
55
95
130
Figure S2l
95
IB:STAT1
IB:pY-STAT1
IB:STAT1
IB:Flag
IB:Tubulin
95
95
130
55
43
55
Figure S2b
For Supplementary Figure 3
IB:Flag
IB:HOIP
IB:Tubulin
130
130
95
55
Figure S3aIB:HA
IB:HOIP
IB:Tubulin
72
130
95
55
Figure S3bIB:HA
IB:Flag
IB:Tubulin
72
13095
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43
55
Figure S3c
130
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IB:p-STAT2
IB:HOIP
IB:Tubulin
Figure S3d IB:HA
IB:Myc
IB:HA
IB:Tubulin
130180
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180
72
55
Figure S3f
IB:STAT2
130
IB:pY-STAT1
IB:Flag
IB:Tubulin
95
95
55
Figure S3g
IB:pY-STAT1
IB:STAT1
IB:Flag
IB:Tubulin
95
95
130
5543
55
IB:pY-STAT1
IB:Myc
IB:Tubulin
95
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55
Figure S3j
Figu
re S
3k
For Supplementary Figure 4
IB:Myc
IB:Flag
IB:Myc
IB:Tubulin
95
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95
55
Figure S4bIB:Flag
IB:Myc
IB:Flag
IB:Tubulin
95
130
95
95
130
55
130180
95
IB:Linear ub
IB:Myc
Figure S4cIB:pY-STAT1
IB:HOIP
IB:Tubulin
IB:pY-STAT1
IB:Lamb1
95
130
95
55
95
72
Figure S4a
IB:Myc
IB:Tubulin
95
55
IB:STAT1
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IB:STAT1
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IB:HA
IB:STAT1
IB:Flag
IB:Tubulin
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43
55
95130
95
72
Figure S4d
IB:HA
72
IB:STAT1
IB:IFNAR2
IB:Flag
IB:Tubulin
95
72
130
95
55
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55
Figure S4eIB:Myc
IB:HA
IB:Flag
IB:Myc
IB:Tubulin
95
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55
43
95
55
Figure S4g
IB:Myc
IB:HA
IB:Myc
IB:Tubulin
95
72
95
55
Figure S4f
For Supplementary Figure 5
IB:HA
IB:STAT1
IB:HA
IB:Tubulin
130
180
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180
95
55
IB:HA
IB:STAT1
IB:HA
IB:Tubulin
55
180
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130
180
Figure S5aIB:HA
IB:STAT1
IB:HA
IB:Tubulin
130180
95
180
95
55
Figure S5bIB:Linear ub
IB:STAT1
95
130
180
Figure S5c
IB:STAT1
IB:Tubulin
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55
IB:Flag
IB:STAT1
IB:Flag
IB:Tubulin
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95
Figure S5dIB:HA
IB:STAT1
IB:HA
IB:Flag
IB:Tubulin
55
43
95
180
95
130
180
Figure S5eIB:Linear ub
IB:STAT1
IB:Flag
IB:Tubulin
55
43
95
130180
Figure S5fIB:Myc
IB:Flag
IB:Myc
IB:Flag
IB:Tubulin55
43
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43
95
Figure S5g
IB:STAT1
IB:OTULIN
95
43
Figure S5hIB:pY-STAT1
IB:OTULIN
IB:Tubulin
55
43
95
Figure S5i
IB:STAT1
95
IB:Tubulin
55
IB:STAT1
95
For Supplementary Figure 5
For Supplementary Figure 6
55
13095
IB:HOIP
IB:Tubulin
Figure S6aIB:HOIP
IB:STAT1
13095
95
Figure S6bIB:HA
IB:STAT1
IB:HA
IB:Tubulin
55
180
72
95
130
180
Figure S6c
IB:Linear ub
IB:STAT1
95
130
180
Figure S6d IB:HOIP
IB:Tubulin
13095
55
IB:HOIP
IB:Tubulin
95130
55
IB:Linear ub
IB:Myc
IB:Myc
IB:Tubulin
130180
95
95
55
Figure S6e
IB:Linear ub
IB:Myc
IB:pY-STAT1
IB:pY-STAT1
IB:Myc
IB:Tubulin
130180
95
95
95
95
55
Figure S6f
IB:pY-STAT1
IB:Myc
IB:Myc
IB:HOIL-1L
IB:Tubulin
95
95
95
55
55
For Supplementary Figure 7
IB:HOIL-1L
IB:Tubulin
IB:HOIL-1L
IB:Tubulin
IB:HOIL-1L
IB:Tubulin
55
55 55
55
55
55
Figure S7aIB:Linear ub
IB:STAT1
130180
95
Figure S7b
Figure S7c