Extended Data Fig. 5: Analysis the phosphorylation sites of USP20 and its interaction with gp78.
From: Feeding induces cholesterol biosynthesis via the mTORC1–USP20–HMGCR axis
a, Procedure to identify the phosphorylated sites of USP20. b, The top 10 of phosphorylated USP20 peptides. H: high glucose; L: low glucose. c, Mass spectrum showing that USP20 was phosphorylated at S132 and S134. d, Domain organization of USP20. zf-UBP: zinc-finger ubiquitin binding domain; DUSP: domain present in ubiquitin-specific protease. e, Validation of the home-made phospho-specific antibody recognizing p-S132/p-S134 of USP20 (p-USP20). USP20 was immunoprecipitated from HEK 293T cells, treated with or without 1U μl-1 λ-phosphatase and 5U ml-1 CIP (PPase), eluted with FLAG peptide and subjected to immunoblotting. f, Rapamycin attenuates USP20 phosphorylation. WT primary hepatocytes were transfected with the indicated plasmid, pre-treated with 100 nM rapamycin for 30 min, and then stimulated with 25.5 mM glucose plus 10 nM insulin for additional 1 h. Cells were lysed with 0.5% NP-40 containing protease and phosphatase inhibitors and immunoprecipitated with anti-FLAG agarose. g, Eight-week-old male WT mice were subjected to fasting and refeeding. Vehicle or rapamycin (5 mg kg-1) were intraperitoneally injected 1 h before fasting or refeeding respectively. Immunoblot analysis of liver samples was performed. F: fasted; R: refed; V: vehicle; Rapa.: rapamycin. h, i, Serum total cholesterol (h) and triglyceride (i) of the mice in (g) (n = 5 per group). j, k, Liver total cholesterol (j) and triglyceride (k) of the mice in (g) (n = 5 per group). l, In vitro activities of USP20 variants. The inset demonstrated equal amounts of USP20 proteins. m, Immunoblot analysis of endogenous Insig-1 and 2 in WT and gp78-KO CHO cells. n, o, Overexpression of Insig-1 (n) or Insig-2 (o) does not block HMGCR-USP20 interaction. p, Mapping the region of gp78 that interacts with USP20. q, The interaction of gp78 and USP20 is unresponsive to 25-HC and mevalonate. Experiments in (e, f, l–q) were performed as indicated twice with similar results. All values are presented as mean ± SEM. Data were analysed by unpaired two-tailed Student’s t-test (h–k).
