Figure 6

Figure 6

CHIP is required for VBP1-mediated HIF-1α stabilization.A, Interaction between VBP1-Flag and HSP70-HA was analyzed in the HEK293T/17 cells by reciprocal Co-IP as indicated. B, Endogenous HSP70 and CHIP interact with VBP1 in the HEK293T/17 cells. Anti-VBP1 antibody was used for Co-IP. C, Interaction between VBP1-Flag and CHIP-HA was analyzed in the HEK293T/17 cells by reciprocal Co-IP as indicated. D, Overexpression of VBP1 increased the CHIP protein levels. HeLa cells were transfected with the indicated plasmids, and the proteins were detected by Western blotting analysis. E, VBP1 increased the endogenous CHIP protein levels. HeLa cells were transfected with VBP1-Myc, and the proteins were detected by Western blotting analysis. F, Western blotting analysis shows that CHIP protein levels were reduced in the VBP1-silenced HEK293T/17 cell lines. The HEK293T/17 cells were stably transfected with lentiviral pLKO plasmids cloned with specific shRNA targeting VBP1 or control scrambled shRNA. G, VBP1-Flag expression decreased CHIP protein half-life. HeLa cells with ectopic VBP1-Flag expression or control plasmids were treated with 50 μg/ml CHX for indicated time periods before being collected for western blotting analysis. The means and SDs from three independent experiments are shown (lower panel). H, Knockdown CHIP diminished the inhibitory effect on ectopic overexpressed HIF-1α by VBP1 in the HCT116 cells. Cells were transfected with the indicated plasmids, and the proteins were detected by Western blotting analysis. The bands of Flag-HIF-1α (∼110 Kd) and VBP1-Flag (∼25 Kd) were easily distinguished by molecular weight. I, Knockdown CHIP diminished the inhibitory effect on endogenous HIF-1α by VBP1 in cells. HCT116 cells transfected with VBP1-Flag or control plasmids were incubated under normoxia or hypoxia for 12 h, and the proteins were detected by Western blotting analysis. J, HEK293T/17 cells with or without silencing of CHIP expression were transfected with the indicated plasmids and treated with MG132 for 8 h. Cell lysates were immunoprecipitated with anti-Flag antibody. The immunoprecipitates and input were probed for indicated antibodies by immunoblotting. K, Mapping the interaction domain of CHIP with VBP1. GST pull-down assays were performed with the GST or GST fusion proteins containing different domains of the CHIP protein. The whole-cell lysates were prepared from HEK293T/17 cells expressing the Flag-tagged VBP1 protein. HIF-1, Hypoxia-inducible factor-1; HSP, heat shock protein; pVHL, von Hippel-Lindau protein; VBP1, pVHL binding protein 1.