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. 2021 Oct 14;12(10):945. doi: 10.1038/s41419-021-04208-3

Fig. 4. TRIM31 stabilized and activated p53 by inducing the K63 ubiquitination of p53 and inhibiting its K48 ubiquitination by disrupting the MDM2-P53 complex.

Fig. 4

a The MCF7 and ZR-75-30 cells stably interfere with TRIM31 or control, the cell lysates were immunoblotted with anti-TRIM31, anti-p53, and anti-β-actin antibodies. b The qPCR was used to detect the mRNA expression of TRIM31 and P53 in MCF7 and ZR-75-30 cells. c The MCF7 and ZR-75-30 cells stably interfere with TRIM31 or control were treated with cyclohexamide (CHX) for 0, 1, 2 and 4 h, the expression of TRIM31 and P53 were detected by western blot. The line chart was further quantitatively analyzed TRIM31 expression. d The MCF7 cells were co-transfected with HA-TRIM31 and Falg-ub plasmids. The cell lysates were immunoprecipitated by anti-P53 antibody, and then western blot assay with anti-Falg, anti-P53, and anti-HA antibody. e The vitro ubiquitination assay was performed and then the western blot assay with the anti-p53 antibody. f The MCF7 cells were co-transfected with HA-TRIM31, Flag-Ub-K48, and Flag-Ub-K63 plasmids, then the cells lysates were pulled down with P53 antibody and analyzed by western blot. g The MCF7 cells were transfected with HA-TRIM31, Flag-Ub-k48R, and Flag-Ub-K63R plasmids. The cell lysates were immunoprecipitated with anti-P53 antibody and then western blot assay with Flag-tag antibody. h, i The MCF7 cells was transfected with HA-TRIM31 or HA-TRIM31-36A together with Flag-Ub-K63 (h) and Flag-Ub-K48 (i) plasmids. The cell lysates were immunoprecipitated with anti-P53 antibody and then western blot assay with anti-Flag antibody. j The MCF7 cells were co-transfected with HA-P53 and Flag-TRIM31 plasmids, the lysates of cells were immunoprecipitated with anti-HA beads and then western blot assay with anti-Flag, anti-HA, and anti-MDM2 antibody. k Pull-down assay was performed to detect the interaction of TRIM31 and p53 and the proteins were immunoblotted with anti-His, GST antibody. l, m The MCF7 and ZR-75-30 cells stably interfere with TRIM31 or control, the real-time PCR assay of the mRNA expression of TRIM31, P53, P21, and BAX in MCF7 and ZR-75-30 cells (l). Western blot assay of the expression of TRIM31, P53, P21, and BAX protein in MCF7 and ZR-75-30 cells (m). n, o The MCF7 and ZR-75-30 cells stably overexpress TRIM31 or control, the real-time PCR assay of the mRNA expression of TRIM31, P53, P21 and BAX in MCF7 and ZR-75-30 cells (n). Western blot assay of the expression of TRIM31, P53, P21 and BAX protein in MCF7 and ZR-75-30 cells (o). Data are shown as the mean ± SD of at least three independent experiments, and the significant level was identified by *P < 0.05, **P < 0.01, and ***P < 0.001.