Anti acetyl histone h4 lys8

Cells were cultured on 150 mm and fixed with 1% formaldehyde (10 min, RT), followed by 200 mM Glycine for 10 min. The cells were washed twice with ice-cold 1 X PBS, and collected in lysis buffer (1 × PBS containing protease inhibitor cocktail (PIC) and 0.5 mM PMSF). Chromatin preparations were sheared using ChIP-IT Express Enzymatic Shearing Kit according to manufacturer's protocols (Active Motif, Carlsbad, CA, USA). Immunoprecipitations were performed using Magna ChIP A/G Kit according to manufacturer's protocol (Millipore). The following antibodies were used for immunoprecipitation: anti-SMAD3 (E.980.9) (Thermo Fisher); anti-acetyl histone H4 (Lys8) (Millipore); anti-p53 (DO-1); mouse IgG (Santa Cruz, Dallas, TX, USA). Quantitative PCR was used to quantify the immunoprecipitated DNA with the following NOX4 promoter-specific primers: (R1) forward: 5′- AAGGGCATAAGGACCTCTCC-3′, reverse: 5′- AGGGAAAAGTGGTCCAAAG-3′; (R2) forward: 5′- CTGAATCAGATGATGGTCTACACTTG-3′, reverse: 5′- GGTCCAAAGGCTTAACATTC-3′; (R3) forward: 5′- AAGGGCATAAGGACCTCTCC-3′, reverse: 5′- GACTCATTCTCATTTCTAC-3′. The data are represented as fold change over IgG control antibody (Ct IP/Ct IgG). The Ct values were normalized to the input (1%) of sheared chromatin DNA. The data are represented as fold change above background = 2^(^−ΔΔCt).