Fig. 5: USP26 deubiquitinates and stabilizes SIRT1.
a The relative protein levels of SIRT1 and USP26 in different HCC cell lines. b The relative protein levels of SIRT1 in Huh7 cells transfected with the indicated doses of USP26. c. U2OS cells stably expressing GFP-USP26 were immunostained for endogenous SIRT1. Scale bar: 10 μm. The relative fluorescence intensity of SIRT1 in cells with or without GFP-USP26 expression was quantified in the same visual fields. d The protein levels of SIRT1 in USP26 knockout (KO) HCCLM3 cells. e The SIRT1 protein levels in liver tumors from Usp26+/Y and Usp26−/Y mice generated through the strategy described in Fig. 3a. f The protein levels of SIRT1 in USP26-knockdown and re-expressed shRNA-resistant USP26 MHCC97H cells. g The half-life of SIRT1 was measured by adding CHX to HEK293T cells transfected with wild-type (WT) USP26 or mutant USP26 (C304S). h The half-life of SIRT1 in USP26-knockdown and re-expressed shRNA-resistant USP26 MHCC97H cells was measured after treatment with CHX. i. HCCLM3 cells stably expressing SIRT1 were transfected with USP26 and then treated with MG132 (10 μM). Endogenous polyubiquitinated SIRT1 was detected by Western blotting. j, k USP26 KO HEK293T cells were co-transfected with HA-Ub, Myc-SIRT1, USP26 or mutant USP26 as indicated and then treated with MG132. Finally, the cells were lysed and subjected to a ubiquitination assay. l, m Ubiquitinated Myc-SIRT1 was purified from HEK293T cells, followed by incubation with bacteria-purified WT or mutant USP26 for an exogenous ubiquitination assay. The reaction buffer was subjected to immunoblotting or stained with Coomassie blue. n–q Similar to j, K29 and K48 ubiquitination assays were carried out. The red stars (*) in f indicate the positions of USP26. Each graph presents the mean ± SEM. Each blot data is representative of three independent experiments. Data in (c) n = 42 or 48 cells for WT or USP26 overexpressing group from three independent experiments. Data in (e) n = 7 mice per group. Statistical significance was calculated by (b, d, k, m, o, q) one-way ANOVA; (c) two-tailed unpaired t-test; (g, h) two-way ANOVA. Source data are provided as a Source Data file.
