Fig. 4

Hypoxia-induced SKA3 interacted with HIF-1a and hindered its degradation in a UPS-dependent manner. A Western bolt analysis was used to detect the expression of HIF-1a in SKA3 knockdown and overexpression cells under hypoxic conditions. B Immuno-fluorescence technique (IF) verified the co-location between SKA3 and HIF-1a. C Co-immunoprecipitation with anti-SKA3, anti-HIF-1a, and anti-IgG was performed to identify the binding between SKA3 and HIF-1a under hypoxic and normoxic conditions. D Cells with SKA3 knockdown or overexpression were treated with cyclohexamide (CHX) for indicated time to determine the half-life of HIF-1a protein. E Western blotting analysis was used to detect the expression of SKA3 and HIF-1a under normoxic (N) or hypoxic conditions (H) with or without MG132 in SKA3 knockdown and overexpression CCA cells. F-G Lysates from SKA3 knockdown and SKA3 overexpression CCA cells treated with MG132 and transfected with HA-Ub or HA-Ub lys48 alone under hypoxic conditions, and then the level of ubiquitinated HIF-1a was analysed