SIRT1 deacetylates RORγt. (A) Flag-RORγt immunoprecipitated from 293T cells expressing p300 and RORγt (black bars), or p300, RORγt, and SIRT1 (red bars) was analyzed by mass spectrometry, identifying the indicated acetylated lysines and changes in their respective MS1 precursor ion abundances. (B) Structural model of acetylated RORγt with DNA. The N-terminal DNA-binding domain of RORγt with DNA is shown. The blue globules indicate side chains of the K69, K81, and K99 residues that are strongly acetylated by p300 and deacetylated by SIRT1. (C and D) Acetylation of endogenous RORγt immunoprecipitated from thymocytes (C) and Th17 cells (D) was determined by Western blot. (E) MS/MS spectrum of acetylated peptide containing Kac-99 from RORγt. (F) Targeted MRM-HR showing the protein level of RORγt in Th17 cells (top panel, nonacetylated peptide ELFSTDVESPEGLSK, m/z at 819.40²⁺), as well as potential RORγt acetylation level changes at residue Kac-99 (bottom, acetylated peptide DSLHAEVQKacQLQQQQQQEQVAK, m/z at 659.09⁺⁺⁺⁺). (G and H) Acetylated tryptic peptides were monitored by MRM-HR, in technical duplicates between WT and Sirt1^−/− pooled mice (10 mice per each genotype), to quantify differences in RORγt acetylation levels in Th17 cells (G) and in thymocytes (H). Peptides assayed for each acetylated lysine were as follows: Kac-69, LQKacCLALGM^oxSR, m/z at 667.85²⁺ (thymocytes only); Kac-81, DAVKacFGR, m/z at 417.73²⁺; Kac-87/88, MSKacKacQR, m/z at 431.23²⁺; and Kac-99, DSLHAEVQKacQLQQQQQQEQVAK, m/z at 878.45⁺⁺⁺ and 659.09⁺⁺⁺⁺. Peak areas were normalized to account for RORγt protein level changes. Significance was assessed using a two-tailed Student’s t test (P < 0.05). Combined data from two (A) independent experiments or representative data from two (D; 3 mice/genotype) and three (C; 10 mice/genotype) independent experiments are shown. *, P < 0.05.