Fig. 4

AURKA interacts with and stabilizes SIN1. A IP assays coupled with mass spectrometry analysis in OE AURKA-Flag-transfected KTC cells. Representative GO term analysis of upregulated and downregulated genes after transfection with shAURKA. Representative GO term analysis of proteins bound to AURKA after IP experiments. B The interaction of AURKA and SIN1 was detected at endogenous levels in TPC-1 cells. Mg132 was added 6 h prior to cell harvest for immunoprecipitation. Western blotting was performed with the indicated antibodies as shown. C The protein expression levels of SIN1 were examined by western blotting in shAURKA-transfected TPC-1 cells and AURKA-overexpression plasmid-transfected KTC cells. D The protein expression of SIN1 in TPC-1 cells treated with 200 μM CHX for different durations was measured by western blotting analysis. E The protein expression levels of SIN1 and P-AKT were examined by western blotting in shAURKA-transfected TPC-1 cells, and Mg132 (20 μM) was added 6 h prior to cell harvesting. F AURKA knockdown increased the endogenous ubiquitination of SIN1. TPC-1 cells with or without AURKA knockdown were treated with Mg132 (20 μM) for 6 h. Then, the total and ubiquitinated SIN1 levels were analysed by western blotting. G Various SIN1 truncations were generated, and their interaction with AURKA was examined by transfection-IP experiments in 293 T cells. Mg132 (20 μM) was added 6 h prior to harvest