Figure. 7.

PKD interferes with Fbxw7-mediated ubiquitination of Aurora kinase A. A) Western blot analysis was performed to show that CRT101-mediated downregulation of Aurora A was proteasome-dependent. B) Half-life of Aurora A protein was measured from the immunoblot of A. C) HeLa cells were synchronized at the start of M phase by nocodazole and preincubated with MG132 for 30 mins. DMSO (control) or CRT101 was added to the culture and the cells were further incubated for 3 hr. Aurora A was pulled down by IP and the protein was analyzed by Western blotting to assess its ubiquitination by CRT101. Downregulation of Aurora A by knockdown of PKD2 and Cdh1 (D) or PKD2 and Fbxw7 (E) was analyzed by Western blotting (top panel). Quantification of Aurora A protein levels was performed by densitometry analysis (bottom panel). F) Western blot analysis depicting that CRT101-mediated downregulation of Aurora A is inhibited by knockdown of Fbxw7. GAPDH was used as loading control. G) A model of regulation of Aurora A protein stability by PKD. The graph represents average of at least three independent experiments with error bars representing SEM (* p<0.5, ** p < 0.01, *** p < 0.001, ns: not significant).