Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc...

31
Regulation of the linear ubiquitination of STAT1 controls antiviral interferon signaling Zuo et al. Supplementary Information

Transcript of Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc...

Page 1: Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc Myc Flag Flag Tubulin 43 55 72 95 I P I n p u t Flag-HOIP + + + + + + + Myc-STAT1

Regulation of the linear ubiquitination of STAT1 controls antiviral interferon signaling Zuo et al.

Supplementary Information

Page 2: Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc Myc Flag Flag Tubulin 43 55 72 95 I P I n p u t Flag-HOIP + + + + + + + Myc-STAT1

Supplementary Figure 1

a

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Flag-HOIP + + + + + +Myc-STAT1 Δ24

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Page 3: Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc Myc Flag Flag Tubulin 43 55 72 95 I P I n p u t Flag-HOIP + + + + + + + Myc-STAT1

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).

Page 4: Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc Myc Flag Flag Tubulin 43 55 72 95 I P I n p u t Flag-HOIP + + + + + + + Myc-STAT1

Supplementary Figure 2

a cS

tat1

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HOIP

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PKR

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virus invasion, 1 hr

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-IFNα +IFNα -IFNα +IFNαCON LUBAC

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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).

Page 6: Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc Myc Flag Flag Tubulin 43 55 72 95 I P I n p u t Flag-HOIP + + + + + + + Myc-STAT1

Supplementary Figure 3

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shHOIP - #1 #2Flag-IFNAR1 - + + +

Tubulin

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Page 7: Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc Myc Flag Flag Tubulin 43 55 72 95 I P I n p u t Flag-HOIP + + + + + + + Myc-STAT1

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).

Page 8: Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc Myc Flag Flag Tubulin 43 55 72 95 I P I n p u t Flag-HOIP + + + + + + + Myc-STAT1

Supplementary Figure 4

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IP Flag

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Page 9: Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc Myc Flag Flag Tubulin 43 55 72 95 I P I n p u t Flag-HOIP + + + + + + + Myc-STAT1

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.

Page 10: Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc Myc Flag Flag Tubulin 43 55 72 95 I P I n p u t Flag-HOIP + + + + + + + Myc-STAT1

Supplementary Figure 5

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Page 11: Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc Myc Flag Flag Tubulin 43 55 72 95 I P I n p u t Flag-HOIP + + + + + + + Myc-STAT1

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).

Page 12: Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc Myc Flag Flag Tubulin 43 55 72 95 I P I n p u t Flag-HOIP + + + + + + + Myc-STAT1

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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180

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

b

STAT1

IP

VSV (h) 0 0 6 12

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IB:

HOIP

TubulinInpu

t

Inpu

t

Myc-STAT1 - WT WT WT YF YF YFSeV (h) 0 0 6 9 0 6 9

IP Myc

Line

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

Line

ar u

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Myc

IP: M

yc

pY-STAT1

pY-STAT1

Short exp.

Long exp.Myc

TubulinInpu

t

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05

1012

16

20

Rel

ativ

e Ifi

t1 m

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Myc-STAT1 WT WT DM YF YF/DMSeV - + + + +

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N.S

SeV - + + + +

0.0

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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).  

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Supplementary Figure 7

a

b c

d

IP IgG

STAT1

Line

ar u

b

Heart

Rbck1 +/+ +/+ +/-STAT1

130

180

IP

IB:

Mouse No. 1 2 1 2

Heart

HOIL-1L

Tubulin

Liver Spleen

1 2 1 2 1 2 1 2

Rbck1+/- Rbck1+/+

Myc-STAT1 - WT WT DM WT DMmIFNβ - - + + + +

Rbck1+/+ Rbck1+/-

pY-STAT1

MycIP: M

yc

Myc

Tubulin

IB:In

put

HOIL-IL

012

406080

100

ifit1

mR

NA ******

mIFNβ - - + + + +

Rbck1+/+ Rbck1+/-

N.S

Myc-STAT1 - WT DM WT DMWT

WT K511/652R

eLentiviral packaging GFP-STAT1

GFP

-STA

T1

95

55

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95

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Rbck1+/- Rbck1+/+ Rbck1+/- Rbck1+/+

Page 15: Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc Myc Flag Flag Tubulin 43 55 72 95 I P I n p u t Flag-HOIP + + + + + + + Myc-STAT1

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). 

Page 16: Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc Myc Flag Flag Tubulin 43 55 72 95 I P I n p u t Flag-HOIP + + + + + + + Myc-STAT1

Figure 1a

Figure 1bIB:Myc

95

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Figure 1d

IB:HOIP

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For Figure 1

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Figure 1eIB:Linear ub

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Figure 1f

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Supplementary Figure 8: Original scans of immunblots.

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For Figure 2

IB:STAT1

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Figure 2a

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Figure 2bIB:pY-STAT1

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Figure 2cIB:pY-STAT1

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Figure 2d

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Figure 2e

IB:VSV-G

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Figure 2l

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For Figure 3

IB:HA

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Figure 3a

IB:HA

IB:JAK1

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Figure 3b

IB:HA

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Figure 3c

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Figure 3eIB:Myc

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Figure 3f

130180 95

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Figure 3g

IB:pY-STAT1

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IB:Tyk2

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Page 19: Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc Myc Flag Flag Tubulin 43 55 72 95 I P I n p u t Flag-HOIP + + + + + + + Myc-STAT1

For Figure 4

IB:JAK1

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Figure 4a

IB:JAK1

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Figure 4b

IB:JAK1

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Figure 4c

IB:STAT1

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Figure 4d72

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Figure 4e

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Figure 4fIB:Flag

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Figure 4g

IB:STAT1

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Figure 4h IB:Linear ub

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Figure 4i

Page 20: Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc Myc Flag Flag Tubulin 43 55 72 95 I P I n p u t Flag-HOIP + + + + + + + Myc-STAT1

For Figure 5

95

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Figure 5a

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Figure 5b

IB:Linear ub

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Figure 5c

IB:Linear ub

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Figure 5d

IB:STAT1

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Figure 5e

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Figure 5f

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Page 21: Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc Myc Flag Flag Tubulin 43 55 72 95 I P I n p u t Flag-HOIP + + + + + + + Myc-STAT1

IB:STAT1

IB:IFNAR2

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Figure 5hIB:pY-STAT1

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Figure 5iIB:STAT1

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Figure 5g

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For Figure 5

Page 22: Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc Myc Flag Flag Tubulin 43 55 72 95 I P I n p u t Flag-HOIP + + + + + + + Myc-STAT1

For Figure 6

IB:HOIP

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Figure 6dIB:HOIP

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Figure 6eIB:Linear ub

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Figure 6f

130180

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Figure 6h

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Figure 6k

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Figure 6j

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Figure 6i

Page 23: Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc Myc Flag Flag Tubulin 43 55 72 95 I P I n p u t Flag-HOIP + + + + + + + Myc-STAT1

For Figure 7

130180

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Figure 7a

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For Figure 8

IB:pY-STAT1

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Page 24: Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc Myc Flag Flag Tubulin 43 55 72 95 I P I n p u t Flag-HOIP + + + + + + + Myc-STAT1

For Supplementary Figure 1

IB:HOIP

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Figure S1cIB:Flag

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Figure S1g

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Figure S1f

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Page 25: Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc Myc Flag Flag Tubulin 43 55 72 95 I P I n p u t Flag-HOIP + + + + + + + Myc-STAT1

For Supplementary Figure 2

IB:pY-STAT1

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Figure S2c

IB:IFIT1

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Figure S2e

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Figure S2l

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For Supplementary Figure 3

IB:Flag

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Figure S3aIB:HA

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Figure S3bIB:HA

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Figure S3c

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Figure S3f

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Figure S3g

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For Supplementary Figure 4

IB:Myc

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Figure S4bIB:Flag

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Figure S4cIB:pY-STAT1

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Figure S4a

IB:Myc

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Figure S4d

IB:HA

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Figure S4eIB:Myc

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Figure S4g

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Figure S4f

Page 28: Supplementary Information Regulation of the linear ...10.1038...10 43 1-9 Δ1-35 Δ-3 24 IP IgG Myc Myc Flag Flag Tubulin 43 55 72 95 I P I n p u t Flag-HOIP + + + + + + + Myc-STAT1

For Supplementary Figure 5

IB:HA

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Figure S5aIB:HA

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Figure S5bIB:Linear ub

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Figure S5c

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Figure S5dIB:HA

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Figure S5eIB:Linear ub

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Figure S5fIB:Myc

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Figure S5g

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IB:STAT1

IB:OTULIN

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Figure S5hIB:pY-STAT1

IB:OTULIN

IB:Tubulin

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43

95

Figure S5i

IB:STAT1

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IB:Tubulin

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IB:STAT1

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For Supplementary Figure 5

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For Supplementary Figure 6

55

13095

IB:HOIP

IB:Tubulin

Figure S6aIB:HOIP

IB:STAT1

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Figure S6bIB:HA

IB:STAT1

IB:HA

IB:Tubulin

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180

72

95

130

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Figure S6c

IB:Linear ub

IB:STAT1

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Figure S6d IB:HOIP

IB:Tubulin

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IB:HOIP

IB:Tubulin

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IB:Linear ub

IB:Myc

IB:Myc

IB:Tubulin

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Figure S6e

IB:Linear ub

IB:Myc

IB:pY-STAT1

IB:pY-STAT1

IB:Myc

IB:Tubulin

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95

95

95

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Figure S6f

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IB:pY-STAT1

IB:Myc

IB:Myc

IB:HOIL-1L

IB:Tubulin

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95

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For Supplementary Figure 7

IB:HOIL-1L

IB:Tubulin

IB:HOIL-1L

IB:Tubulin

IB:HOIL-1L

IB:Tubulin

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55 55

55

55

55

Figure S7aIB:Linear ub

IB:STAT1

130180

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Figure S7b

Figure S7c