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. 2024 Feb 12;15:1280. doi: 10.1038/s41467-024-45520-8

Fig. 4. Shh promotes Hsp90β deubiquitylation through Usp31.

Fig. 4

a Hepa 1–6 cells were treated with CHX, then exposed to 400 pg/μL Shh for 0–36 h. The cell lysates were analyzed by western blot. b Hepa 1–6 cells were treated with 400 or 800 pg/μL Shh for 24 h, protein ubiquitylations were analyzed by western blot. c HSP90β ubiquitylation assays were performed in primary hepatocytes isolated from mice fed with chow/HFFC diet for 16 weeks and analyzed by western blot. The cell lysates were incubated with immunoprecipitated HSP90β and then immunoblotted with anti-ubiquitin to reveal the ubiquitylation levels. d HSP90β ubiquitylation assays were performed in primary hepatocytes treated with 400 pg/μL Shh after Smo knocked down and plasmid Myc-HSP90β and HA-ubiquitin transfection. The cell lysates were incubated with immunoprecipitated Myc and then immunoblotted with anti-HA to reveal the ubiquitylation levels. e Schematic representation of in vitro deubiquitylation assay. The ubiquitylated Myc-HSP90β was immunoprecipitated from HEK293T cells, incubated with the lysates from primary hepatocytes isolated from mice fed with chow/HFFC diet for 16 weeks. f The expression heatmap of DUB genes in Hepa 1–6 cells treated with or without Shh (n = 3 mice per group). g The expression of 14 DUB genes in the livers of mice on a normal chow or HFFC diet for 25 weeks (for Usp1, chow versus HFFC, P = 0.68, for Usp9x, chow versus HFFC, P = 0.99, for Usp15, chow versus HFFC, P = 0.87, for Usp24, chow versus HFFC, P = 0.99, for Usp25, chow versus HFFC, P = 0.99, for Usp27x, chow versus HFFC, P = 0.99, for Usp31, chow versus HFFC, P = 0.034, for Usp32, chow versus HFFC, P = 0.99, for Usp37, chow versus HFFC, P = 0.96, for Usp38, chow versus HFFC, P = 0.99, for Usp45, chow versus HFFC, P = 0.99, for Usp53, chow versus HFFC, P = 0.99, for Otud4, chow versus HFFC, P = 0.99, for Otud6b, chow versus HFFC, P = 0.97). h Usp31 gene expression in Hepa 1–6 cells treated 0–400 pg/μL Shh with or without 20 μM cyclopamine for 24 h (Shh-0 versus Shh-200, P = 0.0040, Shh-0 versus Shh-200, P = 0.0002, Shh-400 versus Shh-400+ cyclopamine, P = 0.0025). i Primary hepatocytes were transfectied with Usp31 RNAi for 48 h, then cells were treated with 400 pg/μL Shh for 24 h, Hsp90β protein level was analyzed by immunoblotting. j HSP90β ubiquitylation assays were performed in primary hepatocytes treated with 400 pg/μL Shh after Usp31 knocked down and plasmid Myc-HSP90βand HA-ubiquitin transfection. The cell lysates were incubated with immunoprecipitated Myc and then immunoblotted with anti-HA to reveal the ubiquitylation levels. Data are presented as mean ± SEM. n = 6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001, NS no significant difference, one-way ANOVA. Source data are provided in the Source Data file.