Fig. 3: TRIM71 promotes ubiquitination and proteasomal degradation of mutant p53s.

a, b Ectopic expression of TRIM71 reduces the mtp53 protein levels in ES-2 and OVCA420 cells. c The proteasome inhibitor MG132 blocks TRIM71-mediated mtp53 degradation in ES-2 cells. MG132 (20 μm) was supplemented for 6 h prior to cell harvest. d, e Knockdown of TRIM71 increases mtp53 protein levels in ES-2 and OVCA420 cells. f CRISPR-Cas9-mediated ablation of TRIM71 elevates p53-S241F protein expression in ES-2 cells. g Overexpression of TRIM71 induces degradation of exogenous mtp53 in the p53/Mdm2 double-knockout MEFs. Combinations of plasmids encoding Myc-TRIM71, p53-S241F, or EGFP as the control were introduced into the MEFs, followed by the IB assay using antibodies as indicated. h, i The mtp53’s half-life is extended upon TRIM71 depletion. The Ctrl-Cas9 and TRIM71-Cas9 cell lines were treated with 100 mg/ml of CHX and harvested at different time points as indicated. The mtp53 protein was detected by the IB assay h, and quantification of p53/GAPDH ratio is shown in the panel I. j, k TRIM71 promotes ubiquitination of mtp53, S241F and R273H. HCT116^p53−/− cells were transfected with combinations of plasmids encoding mtp53s (S241F or R273H), Flag-TRIM71 or His-Ub as indicated, and treated with MG132 (20 μm) for 6 h before harvested for in vivo ubiquitination assay. l, m TRIM71 mediates p53-R273H ubiquitination through K11-, K27-, K29-, and K63-linked ubiquitin chains. HCT116^p53−/− cells were transfected with combinations of plasmids encoding p53-R273H, Flag-TRIM71, and His-Ub or His-Ub-mutants, and treated with MG132 (20 μm) for 6 h before harvested for in vivo ubiquitination assay. Bound and input proteins were detected by IB assays with the indicated antibodies