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

Figure 7.

Figure 7

Phosphorylation of USP20 on Ser334 decreases its activity on TRAF6.A, SMCs from WT and USP20-S334A mice were challenged without (“-”) or with (“+”) IL-1β (1 ng/ml) for 20 min and then solubilized. SMC lysates were subjected to polyubiquitin affinity pull-downs using agarose beads lacking (“C”, control) or containing covalently attached TUBEs. Pull-downs and cognate SMC lysates were subjected to serial immunoblotting for TRAF6, K63-linked polyubiquitin (K63-Ub), as well as phospho-USP20(Ser334) (“p-USP20”) and β-actin (lysates only). B, for TUBEs pull-downs, ubiquitinated TRAF6 smears (TRAF6-Ub; Mr >60) were normalized to densities of the K63-ubiquitin smears in cognate TUBEs pull-downs; these ratios were plotted along with means ± SD of five experiments with independent SMC lines of each genotype. Compared with unstimulated SMCs: ∗p < 0.01 (two-way ANOVA, with Holm-Šídák multiple comparisons test). The gel mobility of molecular weight markers (kDa) is indicated beside each blot panel. SMC, smooth muscle cell; TRAF, TNFR-associated factor; TUBEs, tandem ubiquitin binding entities; USP20, ubiquitin-specific peptidase 20.