Fig. 5

AURKA stabilizes SIN1 by blocking its interaction with CUL4B. A TPC-1 cells were transfected with siRNA targeting various Cullins, and the expression level of SIN1 protein was analysed by western blotting. B The interaction of CUL4B and SIN1 was detected at the endogenous level in TPC-1 cells. Mg132 (20 μM) was added 6 h prior to cell harvest for immunoprecipitation. C 293 T cells were transfected with the indicated plasmids and analysed by western blotting. Increasing CUL4B expression reduced the expression of SIN1. Mg132 (20 μM) was added 6 h prior to harvest. D CUL4B knockdown reduced the endogenous ubiquitination of SIN1. TPC-1 cells with or without CUL4B knockdown were treated with Mg132 (20 μM) for 6 h. Then, the total and ubiquitinated SIN1 levels were analysed by western blotting. E 293 T cells were transfected with the indicated plasmids, and His-IP was performed and analysed by western blotting. The expression of AURKA reduces the binding between SIN1 and CUL4B. Mg132 (20 μM) was added 6 h prior to harvest. F Western blotting validation of the P13K/AKT signalling pathway and the expression of SIN1 in shAURKA-transfected TPC-1 cells, shAURKA-transfected TPC-1 cells and shAURKA/shCUL4B-transfected TPC-1 cells. G 293 T cells were transfected with the indicated plasmids and siRNA, and MYC-IP was performed and analysed by western blotting. Mg132 (20 μM) was added 6 h prior to harvest. H Various SIN1 truncations were generated, and their interaction with CUL4B was examined by transfection-IP experiments in 293 T cells. Mg132 (20 μM) was added 6 h prior to harvest. I Various SIN1 mutants were cotransfected with CUL4B constructs to examine degradation by SIN1. Mg132 (20 μM) was added 6 h prior to harvest