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Figure S1 Figure S1. (A) Screen of USPs for regulating c-Myc stability. Scores of c-Myc protein abundence was derived from two independent experiments. Plotted c-Myc bands intensities were quantified by Odyssey V3.0 software, normalized to Actin protein level. The scores of control empty vector were assigned as 1. c-Myc expression cotransfected with USPs are shown. RPL, relative c-Myc protein level. (B) Measure the stabilization of c-Myc by USP37 using luciferase based protein degradation system (LPDS). Construction of LPDS was indicated. The LPDS-c-Myc was expressed in 293T cells for 36 hours. Transfected cells were lysed. The luciferase activity was measured by Luciferase reporter System (Promega). The protein abundance was determined by the ration of Firefly luciferase/Renilla luciferase. Firefly-Luc c-Myc Renilla-Luc IRES Firefly-Luc c-Myc Renilla-Luc USP37 Renilla-Luc Stabilization B A
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A HA-c-Myc + + + + Flag-Fbw7α - + - + Flag-USP37 - - + + HA-c-Myc Flag-Fbw7α Flag-USP37 EGFP Figure S2 B Myc WT T58A S62A T58/S62A Myc/AA mutant Flag-USP37 EGFP Flag-USP37 - + - + - + - + Figure S2.The regulation of c-Myc by USP37 is independent of Fbw7. (A) The degradation of c-Myc by Fbw7 is blocked by USP37. 293T cells were transfected and protein levels were monitored by immunoblotting. (B) USP37 were co-expressed with wildtype c-Myc, c-Myc T58A, c-Myc S62A or c-Myc T58/S62A in 293T cells and related proteins were detected using Western blotting. (C) Cells were transfected with siRNA against USP37 or Fbw7 as indicated. The protein levels were analyzed using Western blotting. USP37 siUSP37 - + - + siNCsiFbw7 Myc Actin C
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Figure S3 HA-MYCFlag-USP37MERGE A B GFP-USP37 + + + + HA-MAX - + - + Flag-c-Myc - - + + IP:HA WCE IB:GFP IB:Flag IB:GFP IB:Flag IB:HA Figure S3. Colocalization of USP37 and c-Myc in the nucleus. (A) HA-c-Myc and Flag-USP37 were co- expressed into HeLa cells. Transfected cells were treated MG132, fixed, and the USP37 was stained with an anti- Flag antibody while c-Myc with anti-HA antibody. Images were analyzed by confocal microscopy. (B) HA-MAX or Flag-c-Myc were coexpressed with GFP-USP37 in HEK293T cells. Transfected cells were treated with MG132, and the cell lysates were immunoprecipitated using an anti-HA antibody. Co-immunoprecipitated USP37 was detected using an anti GFP-antibody.
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Figure S4 Figure S4. Expression of USP37 in cancers. (A) The expression of USP37 is correlated with CCNB1. Scatter plots show a positive and significant correlation of mRNA expression between USP37 and the c-Myc target gene CCNB1 in lung adenocarcinoma. (B) mRNA expression data sets showing increased USP37 mRNA levels in breast invasive carcinoma(BRCA),but not in prostate adenocarcinoma(PRAD),head and neck squamous cell carcinoma(HNSC), kidney renal clear cell carcinoma(KIRC) or colon adenocarcinoma (COAD) compared to normal tissue. A B NS * ***
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siNC siUSP37 USP37 DAPIMERGE A USP37 Control Antibody absorbed B Figure S5. Specificity of USP37 antibody. (A) H1299 cells were transfected with control or USP37 siRNA. USP37 was examined using immunofluorescence assay. (B) 0.4 g USP37 antibody was incubated with 40 g purified His-USP37 protein for 4 hours. Serial sections of lung cancer samples were stained with control antibody and the antibody absorbed by USP37 protein. Figure S5
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