Gcn5 h 75

The following antibodies were used for western blotting: HDAC3 (GeneTex, GTX113303, Lot 40436), β–Actin [mAbcam 8226] (ABCAM, 20272), ERRα (ABCAM, 16363), HSP90 (Cell Signaling, 4874), Vinculin (Sigma, V9264), Acetylated Lysine Rabbit Polyclonal (Cell Signaling, 9441, Lot 12), UCP1 (R&D, MAB6158), HA Tag (GeneTex, 115044-01), Myc Tag (GeneTex, 21261), V5 Tag (ThermoFisher, R960-25), Pol II Antibody (N-20, Santa Cruz 899 X, Lot K1215), GCN5 (H-75, Santa Cruz 20698, Lot K0112), PGC-1α Mouse mAb (EMD Millipore, 4C.13, ST1202, Lot TE0349482), Anti-Rabbit IgG-HRP (Cell Signaling, 7074), Anti-mouse IgG-HRP (Cell Signaling, 7076), Mouse monoclonal SB62a Anti-Rabbit IgG light chain (HRP) (ABCAM, 99697). The following antibodies were used for ChIP: HDAC3 (ABCAM, 7030, Lot GR121157-6), H3K27ac (ABCAM, 4729, Lot GR251958-1), ERRα (ABCAM, 16363, Lot GR263385-1), NCoR (previously described³⁹ , raised in rabbit against a.a.1944–2453, affinity purified), H3K4me1 (ABCAM 8895), and H3 (ABCAM 1791).

ChIP assay was performed as described (27 (link)). In brief, resting Th cells were cross-linked for 10 min with 1% formaldehyde and lysed by sonication. After pre-clearing with salmon sperm DNA, bovine serum albumin, and Protein Agarose bead slurry (50%), cell extracts were incubated with either rabbit polyclonal Etv5 H-100, p300 N-15, GCN5 H-75 (Santa Cruz), H3ac, H4K16ac, H4K5ac, H4K8ac, or normal rabbit IgG (all from Millipore) overnight at 4°C. The immunocomplexes were precipitated with protein Agarose beads at 4°C for 2 h, washed, eluted and cross-links reversed at 65°C overnight. DNA was purified, resuspended in H₂O and analyzed by qPCR. Primers were described previously (5 (link), 8 (link)). Unless stated otherwise, the percentage input was calculated by subtracting the amount of immunoprecipitated DNA from the IgG control from the amount of immunoprecipated DNA from the specific antibody and normalized against the amount of input DNA.