Figure 4.

PiHL inhibits p53 activity via MDM2-mediated ubiquitination. (A) Half-life of p53 protein in PiHL-silencing (siPiHL) and control (siNC) HCT116 cells was shortened. Immunoblotting assays were used to detect p53 in HCT116 cells without or with the treatment of cycloheximide (CHX; 100 μg/mL). (B) MG132 (20 μM) abolishes the inhibitory effect of PiHL on p53 protein levels. (C) HCT116 cells with or without PiHL overexpression were treated with Act.D (5nM) or 5-FU (500 μM). The p53 and p21 proteins were examined by western blot assays. (D) Lysates were prepared from 293T cells co-transfected with Myc-MDM2 (1.2 μg), FLAG-RPL11 (1.8 μg), HA-GRWD1 (1 μg) and Biotin-PiHL (1 μg) as indicated for 48 h and then immunoprecipitated with anti-FLAG antibody. Immunoprecipitates (IPs) and inputs were immunoblotted with the indicated antibodies. (E) In vitro ubiquitination of p53 MDM2. Recombinant His₆-p53 was incubated with E1, E2 (Ube2d3), ubiquitin, Mg⁺-ATP, FLAG-RPL11, HA-GRWD1, GST-MDM2 and biotinylated PiHL, or control immunoprecipitates were incubated at 37°C for 30 min as indicated. The samples were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. (F) Knockdown of MDM2 attenuates the p53 degradation by PiHL overexpression.