Skip to main content
. 2023 Jun 20;14:3648. doi: 10.1038/s41467-023-39412-6

Fig. 2. The activation of NRF2 pathway by USP25 deficiency.

Fig. 2

a Western blotting analysis of liver proteins from Usp25+/+ and Usp25−/− mice in Fig. 1d. b Quantification of NRF2 target gene expression via qPCR in the liver from the mice (n = 4 per group). c The levels of GSH in the liver from the mice in Fig. 1d. Usp25 WT: 0 h: n = 3 mice, 2 h: n = 5 mice, 4 h: n = 4 mice, 6 h: n = 5 mice, Usp25 KO: 0 h: n = 3 mice, 2 h: n = 4 mice, 4 h: n = 3 mice, 6 h: n = 5 mice. d Western blotting analysis of the indicated proteins in the primary hepatocytes isolated from Usp25+/+ and Usp25−/− mice (n = 2 biologically independent experiments). e Western blotting analysis of the indicated proteins in HepG2 cells (n = 3 biologically independent experiments). f USP25 promotes ubiquitination of NRF2. USP25 expression in HepG2 cells was knocked down with two independent shRNAs and NRF2 was immunoprecipitated and blotted for the presence of ubiquitin (n = 3 biologically independent experiments). g Stabilization of NRF2 by USP25 depletion. USP25 expression in HepG2 cells was knocked down with two independent shRNAs. The cells were then treated with cycloheximide for various of time before harvested for analyses of the indicated proteins via western blotting (n = 2 biologically independent experiments). The relative expression of NRF2 was plotted. h Overexpression of KEAP1 diminished the effects of USP25 depletion on NRF2. USP25 expression in 293 T cells was knocked down first and the cells were then transiently transfected with KEAP1 or GFP expression plasmids (n = 2 biologically independent experiments). The indicated proteins in these cells were analyzed with western blotting. The numbers under western blotting bands represent normalized relative expression. Error bars denote SEM. Two-tailed student’s t tests analysis (b and c), non-parametric tests with blue colored (b). Source data are provided as a Source Data file.