Fig. 2: TRIM25 decreases p85α stability via ubiquitination.
From: Tripartite motif 25 ameliorates doxorubicin-induced cardiotoxicity by degrading p85α
a Western blot analysis of p85α and TRIM25 levels in HL-1 cells transfected with shRNA-TRIM25 (1 μg/ml) or control shRNA. b Western blot analysis of p85α and TRIM25 in HL-1 cells transfected with TRIM25 overexpression plasmids (TRIM25-WT) (1 μg/ml) or control plasmid. c Representative immunoblot and relative quantification of p85α in HL-1 transfected with shRNA-TRIM25 or shRNA-Ctrl (1 μg/ml), then treated with CHX (100 μg/ml) for up to 8 h. d Representative immunoblot and relative quantification of p85α in HL-1 transfected with control plasmid, TRIM25-WT or TRIM25-2EA mutation plasmids (TRIM25-2EA) (1 μg/ml), then treated with CHX (100 μg/ml) for up to 8 h. e, f Co-immunoprecipitation (CO-IP) assay analyzed the interaction of endogenous TRIM25 and p85α in HL-1 cells. g Western blotting assessed the level of p85α in HL-1 cells treated with or without MG132 (20 μM) for 12 h. h HL-1 cells transfected with the plasmids as indicated and treated with DOX (1 μM) for 12 h. Immunoblot analysis of the cell lysates and p85α-IP with the indicated antibodies. Data represent the means ± SEM. (n = 3). P values were determined by unpaired 2-tailed Student t test (a, b) and two-way ANOVA between multiple groups (c, d). WT, wild-type; ctrl, control; TRIM25-2EA, TRIM25 mutant with Glu9 and Glu10 changed into Ala.
