Figure 5.

OTUD6B interacts with elongin B and enhances the binding of pVHL and elongin C. A) MHCC‐LM3 cells with NC or OTUD6B knockdown were treated with MG132 for 8 h. The lysates were subject to immunoprecipitation with control IgG or anti‐pVHL antibodies. The immunoprecipitates were then blotted with indicated antibodies. B) HA‐Ub was cotransfected together with indicated constructs in HEK293 cells. Then the lysates were subject to immunoprecipitation with anti‐flag antibody. The immunoprecipitates were then blotted with anti‐HA antibody. C) MHCC‐LM3 Cells with NTC or OTUD6B knockdown were treated with MG132 for 8 h, and then lysed in 0.5 mL of CHAPS lysis buffer. 500 µL of the lysate was loaded onto a Superdex 200 10/300 GL column. Chromatography was performed on the AKTA‐FPLC with CHAPS buffer. One column volume of eluates was fractionated with 500 mL in each fraction, at the elution speed of 0.5 mL min⁻¹. 30 μL aliquots of each fraction were loaded onto SDS–PAGE gels and detected with indicated antibodies. D) The purified his‐OTUD6B was incubated respectively with purified GST or GST‐elongin B. The mixtures were subjected to GST pull down and blotted. E) HCC cell lysates were subject to immunoprecipitation with control IgG or anti‐OTUD6B antibodies. The lysates and immunoprecipitates were then blotted. F) The lysates of HEK293T cells transfected with indicated constructs were subjected to immunoprecipitation with anti‐flag. The immunoprecipitates or the eluates were then blotted. G) Increasing amounts of myc or flag‐OTUD6B were cotransfected together with indicated constructs into HEK293T cells and the lysates were subject to immunoprecipitation with anti‐myc antibody. The lysates and immunoprecipitates were then blotted. H) The lysates of HEK293T transfected with indicated shRNA and constructs were subject to immunoprecipitation with anti‐myc antibody. The lysates and immunoprecipitates were then blotted. I) The predicted work model of OTUD6B binding to CBC^VHL complex.