Fig. 5

SKA3 recruited PARP1 to bind to HIF-1a, thus stabilizing HIF-1a in a PAR-dependent manner under hypoxic conditions. A Sensitive silver staining method was used to seek for differential expressed proteins under hypoxic conditions. B IP/MS analysis was used to determine which proteins bind to SKA3, thus identifying PARP1 is an interacting protein under hypoxic conditions. C Co-immunoprecipitation with anti-PARP1, anti-HIF-1a or anti-IgG were performed to identify the binding between PARP1 and HIF-1a. D Western blotting analysis was used to detect the expression of HIF-1a in PARP1 knockdown and overexpression CCA cells under hypoxic conditions. E Lysates from PARP1 knockdown, PARP1 overexpression and PARG inhibitor CCA cells treated with MG132 and transfected with HA-Ub plasmids under hypoxic conditions before collecting were subjected to immunoprecipitation and detected with the indicated antibodies. F Immuno-fluorescence technique (IF) verified that SKA3 bound to PARP1 in nucleus and cytoplasm. G Co-immunoprecipitation with anti-SKA3, anti-PARP1, and anti-IgG were performed to identify the binding between SKA3 and PARP1. H Western bolt analysis was used to detect the expression of PARP1 in SKA3 knockdown and overexpression CCA cells under hypoxic conditions. I Western bolt analysis was used to detect the binding ability between PARP1 and SKA3 in SKA3 knockdown and overexpression CCA cells under hypoxic conditions. J Western blotting analysis was used to detect the binding ability between PARP1 and HIF-1a in SKA3 knockdown and overexpression CCA cells under hypoxic conditions. K Lysates from SKA3 knockdown and overexpression CCA cells treated with MG132 were subjected to immunoprecipitation and detected with the indicated antibodies. L The level of ubiquitinated HIF-1a was detected in SKA3 knockdown, PARGi transfected combined with SKA3 knockdown and normal CCA cells under hypoxic conditions