Mdm2 19058 1 ap

The pCMV-Flag-TRIM31, TRIM31 domain deletion mutant plasmids, pLVX-HA-p53, and the p53 domain deletion mutant plasmids were generated using standard molecular techniques. Flag-Ub, Flag-Ub-K48, Flag-Ub-K63, Flag-Ub-K48R, and Flag-Ub-K63R were kindly provided by Hongbo Hu (Sichuan University, Chengdu, China). The primary antibodies against TRIM31 (#12543–1-AP) and MDM2 (19058-1-AP) were obtained from Proteintech (Chicago, IL, USA). The primary antibodies against HA-Tag (C29F4), Flag-tag (D6W5B), p53 (1C12), p21 (12D1), bax (D2E11) were from Cell Signaling Technology (Danvers, MA, USA). The primary antibodies against ubiquitin and β-actin were purchased from Huabio (Hang Zhou, Ze Jiang, China). The Nutlin3 (548472-68-0) and PFTα (63208-82-2) were purchased from TargetMol (Shanghai, China).

After harvesting cells using RIPA buffer, the lysate was incubated overnight with primary antibodies MDM2 (66511-1-Ig, Proteintech), P53 (10442-1-AP, Proteintech) and Flag (#8146, CST). The protein-antibody complex was incubated with Dynal magnetic beads for 2 h and then boiled with loading buffer for 10 min. The obtained supernatant was used for western blotting analysis. Antibodies from different species were used to eliminate the effects of light and heavy chains of primary antibodies on the analysis. The following antibodies were used in western blotting: P53 (60283-2-Ig, Proteintech), MDM2 (19058-1-AP, Proteintech), and ubiquitin (sc-8017, Santa Cruz).