Figure 7. ACK1 decreases tumor suppressor p53 levels via ECD-dependent p53 protein ubiquitination degradation.

(A, C) SGC-7901 cells were transfected with the indicated concentrations of anti-ACK1 (A) or anti-ECD (B) siRNA, and p53 levels were detected by western blot. (B, D) SGC-7901 cells were transfected with the indicated amount of ACK1 (B) or ECD (D) plasmid, and p53 levels were determined by western blot. (E) SGC-7901 cells were co-transfected with the ACK1 plasmid together with an anti-ECD siRNA, and p53 levels were detected by western blot. (F) SGC-7901 cells were transfected with ACK1 or ECD plasmid prior to exposure to the protein synthesis inhibitor cycloheximide (CHX) for the indicated time. The p53 protein levels were analyzed by western blot (left panel), and the densities of the p53 protein bands at each time point were normalized to β-actin and were calculated into percentages using 100% as the value of the zero time point (right panel). (G-I) SGC-7901 cells were co-transfected with the indicated plasmids or siRNAs for 36h and were then treated with or without 20 μg/ml MG132 for 2 h. The cell lysates were resolved directly by SDS-PAGE or were first immunoprecipitated with HA antibody and the indicated proteins were then detected by western blot.