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. 2023 Jun 11;299(7):104911. doi: 10.1016/j.jbc.2023.104911

Figure 8.

Figure 8

NFκB signaling induced by IL-1βis diminished in USP20-S334A SMCs.A, SMCs from WT and USP20-S334A mice were serum-starved overnight, challenged with the indicated concentration of IL-1β for 20 min (37 °C) and solubilized. Parallel immunoblots of SMC extracts were probed serially with primary IgGs specific for phospho-p65(Ser536) (“phos-p65”), total p65, phospho-USP20(Ser334) (“p-USP20”), total USP20 (designated by the arrow; ∗∗ designates a nonspecific band), and β-actin. B, the band densities for phos-p65 were normalized to cognate band densities for total p65; these ratios were plotted (along with means ± SD) for five independent experiments with independently isolated WT and USP20-S334A SMC lines. Compared with WT: ∗p < 0.05 (two-way ANOVA with Šídák multiple comparisons test). C, SMCs from A were serum-starved overnight, challenged ± IL-1β (1 ng/ml) for 4 h (37 °C), and solubilized. Immunoblots of SMC extracts were probed serially with IgGs specific for VCAM-1, USP20, and β-actin, as in panel A. D, the band densities for VCAM-1 were normalized to cognate band densities for β-actin; these ratios were plotted (along with means ± SD) for four independent experiments with independently isolated WT and USP20-S334A SMC lines. Compared with WT: ∗p < 0.05 (two-way ANOVA with Šídák multiple comparisons test). The gel mobility of molecular weight markers (kDa) is indicated beside each blot panel. IL-1, interleukin-1; SMC, smooth muscle cell; USP20, ubiquitin-specific peptidase 20.