Fig. 3

LSH stabilizes p53 protein levels by disrupting the interaction between p53 and MDM2. a LSH affects MDM2 protein levels. Cell lysates were immunoblotted with monoclonal anti-MDM2 in HK1 cells stably expressing vector or LSH. Representative images from three independent experiments are presented. b LSH affects MDM2 protein levels. Cell lysates were immunoblotted with monoclonal anti-MDM2 in A549 cells stably expressing shControl or shRNA LSH. Representative images from three independent experiments are presented. c LSH affects MDM2 protein levels. Cell lysates were immunoblotted with monoclonal anti-MDM2 in HCT116 cells stably expressing shControl or shRNA LSH. Representative images from three independent experiments are presented. d LSH impacts MDM2 mRNA levels. LSH, MDM2, p53, and p21 mRNA levels were detected by real-time PCR in HK1 cells stably overexpressing FLAG-LSH or vector control and A549 cells stably overexpressed LSH shRNA or control shRNA. Error bars represent the SEM of triplicate experiments. Statistical analysis was performed using Student’s t test. *P < 0.05; **P < 0.01. e A549 cells stably expressing shCon or LSH shRNA were lysed, and the lysates were incubated with IgG or p53 (DO-1) antibody. Protein adsorbed by magnetic beads was blotted with the indicated antibodies. Representative images from three independent experiments are presented. f A549 cells stably transfected with shCon or LSH shRNA were treated with or without doxorubicin (DOX, 1 μM). After 24 h, the cells were harvested and analysed by immunoblotting. The number indicates the ratio of phosphorylated p53 (Ser15) and phosphorylated p53 (Ser20) to the corresponding total p53 protein in the doxorubicin-treated experiments (control set to 1). Quantitation of the intensity of the phosphorylation p53 and total p53 signals is shown in the left panel. N = 3, *P < 0.05; **P < 0.01. g A549 and HCT116 cells transiently overexpressing shCon, MDM2 shRNA, and His-Ub were treated with 50 μM MG132 for 4 h, and then, cell lysates were immunoprecipitated with anti-p53 polyclonal antibodies and immunoblotted with monoclonal anti-p53 (DO-1) antibody or anti-Ub antibody. Representative images from three independent experiments are presented