Fig. 3: USP13 directly binds STAT1 and maintains the stability of STAT1.

a Schematic illustration of the interactomes for USP13 substrate screening. HL-1 (Dataset I) and NRCMs (Dataset II) were transfected with Flag-vector or Flag-USP13 plasmids, followed by Ang II stimulation (1 μM, 24 h). Anti-Flag and protein G-Sepharose beads were added to the cell samples for co-IP. Flag-vector plasmid was used as a negative control to exclude non-specific proteins bound to Flag, anti-Flag and protein G-Sepharose beads. The binding proteins were extracted, digested to peptide, and then subjected to LC-MS/MS analysis. The following table showed the candidate substrates of USP13 screened by interactomes. b, c 2D plots with the log₁₀ signal intensity of the quantified proteins on the y axis (revealing the enrichment in Flag-USP13-IP) and the molecular weight (MW) of proteins on the x axis were identified from Dataset I (b) and II (c). d Co-IP of endogenous USP13 and STAT1 in lysates of Ang II- stimulated HL-1 (1 μM, 24 h). e Co-IP of endogenous USP13 and STAT1 in lysates of Ang II- treated heart tissues (1000 ng/kg/min, 4 weeks). f Co-IP of exogenous Flag-USP13 and STAT1 in lysates from NIH/3T3 expressing Flag-USP13 and STAT1. g Representative western blot of Flag-USP13, P-STAT1 and STAT1 in HL-1 expressing Flag-USP13. h, i Representative western blot of STAT1 and Flag-USP13 in HL-1 expressing Flag-USP13 or Flag-vector with CHX (25 μg/mL) pulse-chase stimulation (h) and the quantitative analysis of STAT1 (i; n = 3 independent experiments, adjusted P values were determined by two-way ANOVA with Bonferroni’s correction). j The mRNA levels of Usp13 and Stat1 in HL-1 expressing Flag-USP13 or Flag-vector (n = 6 independent experiments, P values were determined by two-tailed unpaired t test). For i, j, data are presented as mean ± s.e.m.