Fig. 4: USP13 regulates the stability and deubiquitination of STAT1 at residue K379 via its active site C343.

a HL-1 cells were transfected with Flag-USP13 and then subjected to Ang II (1 μM, 24 h) and MG132 (20 μM, 2 h). Lysates were subjected to Co-IP with anti- STAT1, which was followed by western blot of Ub, Flag-USP13 and STAT1. b NRCMs were transfected with siUSP13 and then subjected to Ang II (1 μM, 24 h) and MG132 (20 μM, 2 h). Lysates were subjected to Co-IP with anti- STAT1, which was followed by western blot of Ub, USP13 and STAT1. c STAT1, HA-Ub, or its mutant reserving only K48 (HA-K48) or K63 (HA-K63) were transfected into NIH/3T3 with or without Flag-USP13 and then subjected to 20 μM MG132 for 2 h. Co-IP assays were performed with anti-STAT1 and followed by western blot of HA, Flag-USP13 and STAT1. d Schematic illustration of USP13 active site (C343). e STAT1 and HA-Ub were transfected into NIH/3T3 with or without Flag-USP13 (WT or C343A) followed by MG132. Co-IP assays were performed with anti-STAT1 and followed by western blot of HA, Flag and STAT1. f Representative western blot of STAT1 and Flag-USP13 in NIH/3T3 expressing Flag-USP13 (WT or C343A) with CHX (25 μg/mL) pulse-chase stimulation and the quantitative analysis of STAT1 (n = 3 independent experiments, adjusted P values were determined by two-way ANOVA with Bonferroni’s correction and data are presented as mean ± s.e.m). g Schematic illustration of the STAT1 ubiquitinated-lysine residue (K379). h STAT1 (WT or K379R) and HA-Ub were transfected into NIH/3T3 with or without Flag-USP13 and followed by MG132. Ubiquitinated STAT1 was enriched with anti-STAT1 and then was detected with HA, Flag and STAT1. i Representative western blot of Flag-USP13 and STAT1 in NIH/3T3 expressing Flag-USP13 and STAT1 (WT or K379R). j Scheme for the mechanism of USP13 deubiquitinates STAT1.