a Atg12 Rab9 (ER) F-USP13 Merge (Autophagy) F-USP13 Merge COX4 - - PDF document

▶
a
SMART_READER_LITE
LIVE PREVIEW

a Atg12 Rab9 (ER) F-USP13 Merge (Autophagy) F-USP13 Merge COX4 - - PDF document

Sep 14, 2023 245 likes •390 views

a Atg12 Rab9 (ER) F-USP13 Merge (Autophagy) F-USP13 Merge COX4 (Mito) F-USP13 Merge Mock HSV-1 b BFP-USP13 BFP-USP13 FLAG-STING Rab9 (ER) FLAG-STING Merge Mock HSV-1 (4h) HSV-1 (8h) Supplementary Figure 1 USP13 and STING colocalize at ER. ( a )

slide-1
SLIDE 1

a

Rab9 (ER) F-USP13 Merge Atg12 (Autophagy) F-USP13 Merge Mock HSV-1 COX4 (Mito) F-USP13 Merge

b

Mock HSV-1 (4h) HSV-1 (8h) Rab9 (ER) BFP-USP13 FLAG-STING BFP-USP13 FLAG-STING Merge Supplementary Figure 1 USP13 and STING colocalize at ER. (a) Immunofluorescence staining (with anti-Atg12, anti-COX4 or anti-FLAG) and confocal microscopy analysis of HeLa cells transfected with Rab9 (an ER marker) and FLAG-USP13 for 20 hours followed by mock infection or HSV-1 infection for 8 hours. (b) Immunofluorescence and confocal microscopy analysis of HeLa cells transfected with Rab9, BFP-USP13 and FLAG-STING for 20 hours followed by HSV-1 infection for 0-8 hours. Arrow heads indicate colocalization of STING and USP13 at ER. Scale bars represent 20 mm. Data are representative of three independent experiments.

slide-2
SLIDE 2

1000 2000 3000 4000

  • Rel. mRNA Level

SeV: 0 4 8 12 20 40 60 80 250 500 750 1000 IFNB TNFA IL6

a b

shCon shUSP13#1 shUSP13#2 h

c

siRNA: N.C. #1 #2 #3

  • USP13
  • b-Actin

d e

siN.C. 0 6 12 siUSP13#2 6 12 THP-1 h

  • pIkBa
  • IkBa
  • pIRF3
  • IRF3
  • USP13
  • b-Actin

siN.C. 0 6 12 siUSP13#2 6 12 THP-1 h SeV: HSV-1:

  • pIkBa
  • IkBa
  • pIRF3
  • IRF3
  • USP13
  • b-Actin

Supplementary Figure 2 Knockdown of USP13 did not affect SeV-triggered signaling. (a) qRT-PCR analysis of IFNB, TNFA, and IL6 in THP-1 cells stably transfected with control shRNA, shUSP13#1 or shUSP13#2, then infected with HSV-1 for 0-24 hours. (b) Immunoblot analysis of phosphorylated and total IRF3, IkBa, ERK, p38, and JNK and b-Actin in THP-1 cells stably transfected with control shRNA or shUSP13#1, then infected with HSV-1 for 0-12 hours. (c) Immunoblot analysis (with anti-USP13 or anti-β-Actin) of THP-1 cells transfected siRNAs targeting USP13 (#1, #2, #3) or non-targeting control siRNA(N.C.) for 36 hours. (d) qRT-PCR analysis of IFNB and TNFA in THP-1 cells transfected with siUSP13#2 or N.C. for 36 hours, then infected with HSV-1 or SeV for 0-8 hours. (e-f) Immunoblot analysis of phosphorylation of IRF3 and IkBa, total IRF3 and IkBa, USP13 and b-Actin in THP-1 cells transfected with siUSP13#2 or N.C. for 36 hours, then infected with SeV (e) or HSV-1 (f) for 0-8 hours. *P < 0.05; ***P <0.001 (analysis of two-way ANOVA followed by Bonferroni post-test). Data are representative of three independent experiments (mean  S.D. in a and d).

f

shCon SeV: 0 4 12 shUSP13#1 THP-1

  • pIkBa
  • IkBa
  • pIRF3
  • IRF3
  • pERK
  • ERK
  • pJNK
  • JNK
  • USP13
  • b-Actin

h 8 4 12 8

  • p65
  • p-p65

IFNB

  • Rel. mRNA Level

siN.C. siUSP13#2 3 6 * *** TNFA 250 500 3000 4000 5000 * 20 30 10 50

  • 50
  • 90
  • 90
  • 34
  • 34
  • 34
  • 50
  • 50
  • 50
  • 50
  • 50
  • 50
  • 50
  • 50
  • kDa

34

  • 50
  • 50
  • 90
  • 50
  • 34
  • 34
  • 50
  • 50
  • 90
  • 50
  • MW (kDa)

MW (kDa) MW (kDa)

slide-3
SLIDE 3

a

Bone marrow

  • b-Actin

Usp13: +/+ m/m

  • USP13
  • NS

120- 90- 50- 34- 26- 50-

c

+/+ +/m m/m

GCCCACGATCCGCGTGC GCCCACGACTCCGCGTG GCCCACGATCCGCGTGC CTCCGCGTG

91 CCC ACG ACT CCG…….CTC TGA 171 bp 31 P T T P …… L * 56aa (early termination) 91 CCC ACG ATC CGC……C TCT GAA CTC….. AGCTAA 2577 bp 31 P T I R …… S E L ....… S * 858aa (full length) Reading frame

b

exon1 exon2 exon3

5’···CATATGCCCACGATCCGCGTGCCCAGGTCGGGGGACCGCGTCT···3’ 3’···GGTGCTAGGCGCACGGGTCCAGC···5’

Usp13 allele

Cas9-gRNA PAM Cas9 gRNA sequence: Wild-type Usp13 allele: 5’···CATATGCCCACGATCCGCGTGCCCAGGTCGGGGGACCGCGTCT···3’ Insertion 5’···CATATGCCCACGACTCCGCGTGCCCAGGTCGGGGGACCGCGTCT···3’ Mutant Usp13 allele: ATG Supplementary Figure 3 Generation of Usp13m/m mice. (a) A scheme for CRIPSR/Cas9-mediated genome editing of the Usp13 gene locus. (b) Gene sequence and reading frame of Usp13+/+, Usp13+/m and Usp13m/m mice. (c) Immunoblot analysis of USP13 in bone marrow cells from Usp13+/+ and Usp13m/m mice. Data are representative of two (c) independent experiments. MW (kDa)

slide-4
SLIDE 4

d e

CD4 CD8 Usp13+/+ Usp13m/m 2.24 2.43 6.79 6.08 79.7 80.9 20 40 60 80 100 Usp13: m/m +/+ Cell number (x 106) Thymus

c

CD3 CD8 CD25 CD44 CD4 CD4 CD62L CD19 Usp13+/+ Usp13m/m 31.2 32.3 53.6 52.9 35.8 34.3 53.9 51.6 1.44 1.36 19.3 19.7 21.4 23.3 55.5 57.6 100 200 300 400 Usp13: m/m +/+ Cell number (x 106) Spleen

f

CD3 CD8 CD25 CD44 CD4 CD4 CD62L CD19 Usp13+/+ Usp13m/m 72.6 66.4 22.6 28.0 48.8 46.3 48.5 47.3 4.32 4.41 31.9 31.6 7.01 7.62 72.9 70.8 2 4 6 8 Usp13: m/m +/+ Cell number (x 106) Peripheral lymph nodes 67.5 68.3 Usp13+/+ Usp13m/m GM-CSF culture CD11c

CD11b

F4/80 M-CSF culture 96.2 97.9 CD11c CD11b SSC B220 Usp13+/+ Usp13m/m Flt3L culture 79.0 78.2 23.3 22.9 66.7 69.1

a

Genotype Usp13+/+ Usp13+/m Usp13m/m Number 121 258 110 Percentage 24.7% 52.8% 22.5%

b

6 7 8 9 12 15 18 21 24 F-Usp13+/+ F-Usp13m/m M-Usp13+/+ M-Usp13m/m Weeks after birth Body Weight (g) Supplementary Figure 4 USP13 deficiency did not alter the homeostasis of immune cells. (a) Mice numbers and percentages of each genotype. (b) Mice body weight of each genotype. F, Female; M, Male. (c) Flow cytometry analysis of GM-CSF, M-CSF or Flt3L induced DCs, Macrophage cells or pDCs from Usp13+/+ and Usp13m/m mice. (d-f) Flow cytometry analysis of immune cells and quantitative data in thymus (e), spleen (f) and peripheral lymph nodes from Usp13+/+ and Usp13m/m mice. Data are representative of two (c-f) independent experiments (n=3) (mean  S.D. in b, d-f).

slide-5
SLIDE 5

MEFs WT Usp13m/m 4 8 4 8 HSV-1: h

  • β-Actin
  • p-IRF3
  • USP13
  • IRF3

a

MEFs 0.082 0.041 3.62 2.29 GFP SSC WT Usp13m/m Mock HSV-1-GFP

b c

7.68 5.27 MOI=0.05 MOI=0.1

d

MEFs WT Usp13m/m 4 8 4 8 SeV: h

  • β-Actin
  • p-IRF3
  • USP13
  • IRF3

e

HSV-1 UL30 Rel.mRNA Level 100 200 300 20000 30000 40000 50000 *** *** Usp13+/+ Usp13m/m Usp13+/+ Usp13m/m MEF-Ifnb MEF-Ifna4 MEF-Tnf MEF-Ccl5 500 4000 8000

  • Rel. mRNA Level

200 2000 4000 50 100 150 100 1000 2000 *** *** * *** *** *** * *** *** * *** *** ** * Supplementary Figure 5 USP13 deficiency potentiated HSV-1- but not SEV-triggered signaling in MEFs or BMDMs. (a) qRT-PCR analysis of Ifnb, Ifna4, Tnf or Ccl5 mRNA in Usp13+/+ and Usp13m/m MEFs or BMDMs left uninfected (Mock) or infected with HSV-1, SeV, HCMV or EMCV for 0-8 hours, or mock transfected (Lipo) or transfected with ISD45, DNA90 or poly(I:C) for 6 hours. (b) Immunoblot analysis of phosphorylation of IRF3 and IkBa, total IRF3 and IkBa, USP13 and β-Actin in Usp13+/+ and Usp13m/m MEFs infected with SeV or HSV-1 for 0-8 hours. (c) Flow cytometry analysis of Usp13+/+ and Usp13m/m MEFs infected with HSV-1-GFP for 24 hours. Numbers adjacent to outlined areas indicate the percentages of GFP+ MEFs. (d) qRT-PCR analysis of HSV-1-UL30 mRNA in Usp13+/+ and Usp13m/m MEFs infected with HSV-1 for 1 hour followed by twice PBS wash and subsequent incubation in full medium for 0-24 hours. (e) Viral plaque assay of HSV-1 in Usp13+/+ and Usp13m/m MEFs infected with HSV-1 for 1 hour followed by twice PBS wash and cultured in full medium for 32 hours. *P < 0.05; **P < 0.01; ***P <0.001 (analysis of two-way ANOVA followed by Bonferroni post-test). Data are representative of three (a, b) or two (c-e) independent experiments (mean  S.D. in a, d-e).

  • IkBa
  • pIkBa
  • IkBa
  • pIkBa

0.5 1.0 1.5 2.0 2.5 HSV-1 titer (x 104 PFU/ml) Usp13+/+ Usp13m/m BMDM--Tnf BMDM-Ifnb 1000 2000 3000

  • Rel. mRNA Level

*** *** *** ** * BMDM-Ifna4 100 200 300 400 *** *** *** * ** 100 200 250 ** *** ** ** BMDM-Ccl5 400 800 1000 *** *** *** *** ** * 34

  • 34
  • 50
  • 50
  • 90
  • 50
  • 34
  • 34
  • 50
  • 50
  • 90
  • 50
  • MW (kDa)

MW (kDa)

slide-6
SLIDE 6

FLAG-USP13

  • MAVS
  • cGAS
  • IRF3
  • STING

Lysate

  • USP13
  • TBK1
  • RIG-I
  • MAVS
  • cGAS
  • IRF3
  • STING
  • USP13

IP: aFLAG IB: aHA IB: aFLAG HA-Ubiquitin cGAS STING IRF3 FLAG- USP13: Denature-IP: aFLAG IB: aHA IB: aFLAG Lysate

  • IgH
  • Ub-modification
  • STING
  • cGAS
  • IRF3
  • STING
  • cGAS
  • IRF3
  • USP13

a c d b

IP: aFLAG IB: aHA IB: aFLAG Lysate IB: aFLAG IB: aHA FLAG-AMFR+HA-STING

  • USP13/USP13(AE)
  • FLAG-AMFR
  • HA-STING

IB: aUSP13

  • FLAG-AMFR
  • HA-STING

IP: aFLAG IB: aHA IB: aFLAG Lysate IB: aFLAG IB: aHA FLAG-TRIM32+HA-STING

  • USP13/USP13(AE)
  • FLAG-TRIM32
  • HA-STING

IB: aUSP13

  • FLAG-TRIM32
  • HA-STING

Supplementary Figure 6 USP13 functions at the level of STING. (a) Immunoprecipitation (with anti-FLAG) and immunoblot analysis (with anti-FLAG, anti-HA or anti-USP13) of HEK293 cells transfected with plasmids encoding FLAG- tagged AMFR or TRIM32, HA-STING and USP13 or USP13 enzymatic inactive mutation for 24 hours. (b) Immunoprecipitation (with anti-FLAG) and immunoblot analysis (with anti-FLAG or anti-HA) of HEK293 cells transfected with plasmids encoding FLAG-USP13 and HA-tagged RIG-I, MAVS, cGAS, STING, TBK1 or IRF3 for 24 hours. (c) Denature-immunoprecipitation (with anti-FLAG) and immunoblot analysis (with anti-FLAG, anti-HA or anti-USP13) of HEK293 cells transfected with plasmids encoding HA-ubiquitin, FLAG-tagged cGAS, STING or IRF3, and either empty vector or USP13 for 24 hours. (d) Luciferase assay analyzing ISRE promoter activity in HEK293 cells transfected with plasmids encoding MAVS, cGAS, STING, TBK1 or IRF3, an ISRE firefly luciferase reporter (ISRE-Luc) and the empty vector, USP13 or USP13(AE) for 24 hours (left graph). Luciferase assay analyzing ISRE promoter activity in HEK293 cells transfected with plasmids encoding RIG-I CARD, STING, TBK1 or IRF3, an ISRE firefly luciferase reporter (ISRE-Luc) and either the empty vector or shRNA targeting USP13 for 24 hours (right). Data are representative of two independent experiments (mean  S.D. in d). + + +

  • ISRE

50 100 150 200 250 Vec shUSP13

  • Rel. Lucif. Act.

ISRE 50 100 150 200 1000 2000 3000 4000 Vec USP13 USP13(AE)

  • Rel. Lucif. Act.

34

  • 34
  • 50
  • 50
  • 120
  • 90
  • 50
  • 120
  • 90
  • 50
  • 90
  • 50
  • 90
  • 90
  • 50
  • 34
  • 50
  • 90
  • 34
  • 50
  • 34
  • 50
  • 90
  • MW (kDa)

MW (kDa) MW (kDa) MW (kDa)

slide-7
SLIDE 7

a

Vec cGAS+STING

b

HEK293-IFN-b Supplementary Figure 7 The isopeptidase and the UBA domains of USP13 deubiquitinate STING. (a) Reporter assays of HEK293 cells transfected with the indicated truncates of USP13 together with the IFN-b luciferase reporter for 24 hours. (b) Denature-IP (with anti-HA) and immunoblot analysis (with anti-FLAG or anti-Myc) of HEK293 cells transfected with plasmids encoding Myc-Ubiquitin, HA-STING, and FLAG-tagged USP13 or USP13 truncates for 20 hours. (c) Schematic structures showing USP13 truncates interacting with STING, inhibiting cGAS and STING-mediated activation of IFN-b promoter and deubiquitinating STING. Data are representative of two independent experiments (mean  S.D. in a). 50 100 150

  • Rel. Lucif. Act.

FLAG-USP13 Lysate FLAG-USP13: Denature-IP: aHA IB: aMyc IB: aHA HA-STING+Myc-Ub

  • STING-Ub
  • HA-STING
  • FLAG-USP13 & truncates
  • HA-STING

c

STING binding 863 + + +

  • +
  • 1

UBP UBA1/2 1 301 625 301 624 300 1 624 Inhibition of IFN-b promoter + +

  • Deubiquitinating STING

+ +

  • 50
  • 50
  • 90
  • 90
  • 34
  • 34
  • 120
  • 34
  • MW (kDa)
slide-8
SLIDE 8

THP1 Denature-IP: aSTING IB: aUb

  • Ub-STING
  • Ub-STING
  • STING

IB: aSTING (Weak Exp) (Strong Exp) SeV Mock shUSP13: + +

  • Lysate
  • STING
  • USP13
  • b-Actin

a b c

MEFs Denature-IP: aSTING IB: aUb

  • Ub-STING
  • STING

IB: aSTING HSV-1 Mock Usp13: m/m m/m +/+ +/+ Lysate

  • STING
  • USP13
  • b-Actin

MEFs Denature-IP: aSTING IB: aUb

  • Ub-STING
  • STING

IB: aSTING SeV Mock Usp13: m/m m/m +/+ +/+ Lysate

  • STING
  • USP13
  • b-Actin

Supplementary Figure 8 USP13 did not regulate SeV-induced ubiquitination of STING. (a) Immunoprecipitation (with anti-HA) and immunoblot analysis (with anti-FLAG, anti-HA, or anti-USP13) of HEK293 cells transfected with plasmids encoding FLAG-STING, HA-Ubiquitin and USP13 or USP13(AE) for 24 hours. (b) Denature-immunoprecipitation (with anti-STING) and immunoblot analysis (with anti-Ub, anti-STING, anti-USP13 or anti-b-Actin) of Usp13+/+ and Usp13m/m MEFs infected for 8h with HSV-1. (c) Immunoprecipitation (with GST beads and TUBE) and immunoblot analysis (with anti-STING, anti-Ub, anti-USP13, anti-b-Actin) of Usp13+/+ or Usp13m/m BMDCs infected with HSV-1 for 0-8 hours. (d-e) Denature-immunoprecipitation (with anti-STING) and immunoblot analysis (with anti-Ub, anti-STING, anti-USP13 or anti-b-Actin) of Usp13+/+ and Usp13m/m MEFs (d) or USP13-knockdown-THP-1cells (e) infected for 8h with SeV. Data are representative of three independent experiments.

d e

FLAG-STING HA-Ubiquitin USP13 USP13(AE) + + + + + + + + IP: aHA IB: aHA IB: aFLAG Lysate

  • FLAG-STING
  • USP13 & USP13(AE)
  • Ub-STING
  • Ub

BMDCs IB: aUb IB: aSTING HSV-1 Mock Usp13: m/m m/m +/+ +/+ Lysate

  • STING
  • USP13
  • b-Actin
  • Ub
  • Ub-STING

Weak Exp.

  • Ub-STING

Strong Exp. Pulldown: TUBE Pulldown: TUBE 50

  • 90
  • 90
  • 34
  • 34
  • 120
  • 50
  • 50
  • 90
  • 90
  • 34
  • 50
  • 120
  • 50
  • 90
  • 120
  • 50
  • 90
  • 120
  • 50
  • 90
  • 120
  • 50
  • 90
  • 120
  • 34
  • 90
  • 50
  • 90
  • 90
  • 34
  • 50
  • 120
  • 50
  • 90
  • 120
  • 34
  • 50
  • 90
  • 90
  • 34
  • 50
  • 120
  • 34
  • MW (kDa)

MW (kDa) MW (kDa) MW (kDa) MW (kDa)

slide-9
SLIDE 9

IP: aFLAG IB: aHA IB: aFLAG Lysate IB: aFLAG IB: aHA FLAG-TBK1 + HA-MAVS

  • USP13/USP13(AE)
  • FLAG-TBK1
  • HA-MAVS

IB: aUSP13

  • FLAG-TBK1
  • HA-MAVS

IP: aFLAG IB: aHA IB: aFLAG Lysate IB: aFLAG IB: aHA FLAG-TBK1 + HA-STING

  • USP13/USP13(AE)
  • FLAG-TBK1
  • HA-STING

IB: aUSP13

  • FLAG-TBK1
  • HA-STING

c a

  • Ub-STING

Eluted STING-Ubi

  • STING
  • USP13

K27O K29O K33O K27R K29R K33R Expression of FLAG-STING and HA-Ub or Ub-mutants Pull down with anti-FLAG agarose Elute with FLAG peptide In vitro translated USP13 Deubiquitination assay (1 mM ATP) IB: aHA IB: aFLAG IB: aUSP13 HA-Ub:

b

Supplementary Figure 9 USP13 deconjugates K37/K33-linked polyubiquitin chains from STING. (a) In vitro deubiquitination of Ubiquitin-modified STING by USP13. (b) Immunoblot (upper panels) and qRT-PCR (lower graphs) analysis of Usp13+/+ and Usp13m/m MEFs that were transfected with control siRNA or siRNA targeting Amfr for 36 hours and then left untreated or infected with HSV-1 for 4-8 hours. (c) Immunoprecipitation (with anti-FLAG) and immunoblot analysis (with anti-FLAG, anti-HA or anti-USP13) of HEK293 cells transfected with plasmids encoding FLAG-TBK1, HA-STING, HA-MAVS and the empty vector (Control), USP13 or USP13(AE) for 20 hours. *P < 0.05; **P < 0.01; ***P <0.001 (analysis of two-way ANOVA followed by Bonferroni post-test). Data are representative of three independent experiments (mean  S.D. in c).

  • Amfr
  • β-Actin

Amfr siRNA Con #1 #2 #3 500 1000 1500 Rel.mRNA Level 20 40 60 Ifnb Tnf HSV-1: ** *** ** *** 50 100 1200 1600 2000 Ccl5 20 40 60 Amfr 4 8 4 8 Usp13+/+ + siCon Usp13+/+ + siAmfr#2 Usp13m/m + siCon Usp13m/m + siAmfr#2 h * *** *** *** ** Rel.mRNA Level 50

  • 34
  • 50
  • 90
  • 90
  • 120
  • 90
  • 90
  • 90
  • 90
  • 90
  • 90
  • 50
  • 90
  • 90
  • 50
  • 120
  • MW (kDa)

MW (kDa) MW (kDa) MW (kDa)

slide-10
SLIDE 10

Supplementary Figure 10 A model on USP13-mediated regulation of antiviral responses. In unstimulated cells, USP13 interacts with and catalyzes deubiquitination of STING which constitutively undergoes K27-linked ubiquitination mediated by AMFR/gp78, thereby restricting the basal immune signaling. Upon infection with DNA viruses, the majority of USP13-interacting STING is disassociated from USP13 and translocates to form puncta, thereby leading to increased ubiquitination of STING and boosted recruitment of TBK1. Meanwhile, a portion of USP13 still interacts with STING to prevent excessive immune and inflammatory responses.

slide-11
SLIDE 11

Figure 1

a b

  • 120
  • 90
  • 50
  • 34
  • 26

90- 50- 34-

C

  • 120
  • 90
  • 50
  • 34
  • 26
  • 20

d

  • 120
  • 90
  • 50
  • 34
  • 50
  • 34

e

  • 120
  • 90
  • 50
  • 34
  • 50
  • 34
  • 120
  • 90
  • 50
  • 34
  • 50
  • 34
  • 120
  • 90
  • 120
  • 90
  • 120
  • 90
  • 50
  • 34
  • 26
  • 20

Supplementary Figure 11 Original immunoblots for Figure 1

slide-12
SLIDE 12

Figure 2

c

Figure 3

c

  • 50
  • 34
  • 34
  • 90
  • 50
  • 50
  • 50
  • 90
  • 120

Figure 5

a

  • 34
  • 90
  • 50
  • 50
  • 50
  • 120
  • 34
  • 90
  • 120
  • 50
  • 34
  • 90
  • 50
  • 50
  • 50
  • 120
  • 34
  • 50
  • 50
  • 90
  • 120
  • 90
  • 120

a

Supplementary Figure 12 Original immunoblots for Figures 2, 3, and 5

slide-13
SLIDE 13

Figure 6

a

Figure 7

c d

  • 50
  • 120
  • 50
  • 34
  • 120
  • 50
  • 50
  • 90
  • 120
  • 90
  • 26
  • 90
  • 34
  • 50
  • 34
  • 120
  • 34
  • 26
  • 120
  • 50
  • 50
  • 90
  • 120
  • 90
  • 90
  • 34
  • 50
  • 34

a

  • 34
  • 120
  • 90

b c

  • 50
  • 120
  • 90
  • 50
  • 120
  • 90
  • 50
  • 120
  • 90
  • 50
  • 34
  • 120
  • 90
  • 50
  • 34

d e

  • 50
  • 120
  • 90
  • 50
  • 34
  • 50
  • 34
  • 50
  • 34
  • 50
  • 120
  • 90
  • 50
  • 50
  • 34
  • 50
  • 34
  • 50
  • 120
  • 90
  • 50
  • 34
  • 120
  • 90
  • 50
  • 34
  • 50
  • 120
  • 90
  • 50
  • 34
  • 50
  • 34

a b

  • 120
  • 90
  • 120
  • 90
  • 34
  • 90
  • 120
  • 50
  • 120
  • 90
  • 120
  • 90
  • 50
  • 34

Supplementary Figure 13 Original immunoblots for Figures 6 and 7