Anti acetylated histone h3 antibody

ChIP assays were performed as previously described^{11 (link)}. Briefly, formaldehyde-fixed chromatin was subjected to sonication to obtain DNA fragments ranging in size from 100 to 400 bp. Chromatin fragments were immunoprecipitated using anti-acetylated histone H3 antibody (Millipore, #06-599), or anti-acetylated histone H4 antisera (Millipore, #06-866), or rabbit control Immunoglobulin (DAKO). After reversal of formaldehyde crosslinks, precipitated DNA was analyzed by quantitative real-time PCR using primers as follows: Il17a promoter, 5′-CACCTCACACGAGGCACAAG-3′ and 5′-ATGTTTGCGCGTCCTGATC-3′; Il17f promoter, 5′-GGCTGCTTCTTCCCTCAGG-3′ and 5′-TAAAACTGACAGGTACTACTGC-3′; Il21 promoter, 5′-CTGCAATGGGAGGGCTTGG-3′ and 5′-CTTCAACCTGACTGTGCACAG-3′; Il23r promoter, 5′-CAAGAGTCCTTAAAACCCACC-3′ and 5′-CATGGGAAGTGGCATTATTAGG-3′; Rorc (RORγt) promoter, 5′-CAGAAACACTGGGGGAGAGC-3′ and 5′-ACACAGCTGGCAGTGGAGG-3′; Rora (RORα isoform 4) promoter, 5′-GCAAGGCAGAGAGCTTCCG-3′ and 5′- CACCAAAGTCCCTCGCCAC-3′; Il5 3′-end, 5′-ATGAGAGGATGAATGAATGAATG-3′ and 5′-AGCTCTTCATCCTTGTACAGC-3′; Actb promoter, 5′-CTGTGGCGTCCTATAAAACCC-3′ and 5′-CGAAGGAGCTGCAAAGAAGC-3′.

Whole-cell lysates from control and HDACi-treated subconfluent cell cultures or crushed frozen tumours were prepared in ice-cold RIPA buffer [10 mM Tris-HCl (pH 7.4), 5 mM EDTA (pH 8), 1% (v/v) Nonidet P-40, 0.5% (v/v) sodium deoxycholate, 0.1% (v/v) SDS] supplemented with protease inhibitor cocktail (Roche, cat. #04693132001). Lysates were sonicated four times for 30 s, cleared by centrifugation at 13,000 g for 15 min at 4°C and the supernatants stored at −20°C.
Western blotting was performed using standard methodology as described previously (Denoyer et al., 2014 (link)). Briefly, 40 µg of proteins were separated on a 15% SDS-PAGE gel and transferred to a PVDF membrane. The membrane was blocked with 10% (w/v) non-fat dried milk in PBS containing 0.05% Tween-20 for 1 h at room temperature and incubated with anti-acetylated histone H3 antibody (Millipore, cat. #06-599, 1:10,000 dilution) overnight at 4°C. After three washes with wash buffer (0.025% Tween-20 and 0.1% BSA in PBS) for 10 min, the membrane was incubated for 1 h with an appropriate horseradish-peroxidase-conjugated secondary antibody in wash buffer. Specific protein bands were detected using enhanced ECL reagents and Super RX film or ChemiDoc™ MP System (Bio-Rad). An anti-GAPDH antibody (Abcam, cat. #Ab8245, 1/10,000 dilution) was used as loading control.