Anti h4k12ac

Cells were lysed with RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40) in the presence of protease and phosphatase inhibitors. For subcellular fractionation, 2 × 10⁸ cells were collected for the extraction and both NE-PER nuclear and cytoplasmic extraction kits (Thermo-Scientific, Waltham, MA) were used. Prior to SDS-PAGE, cell lysates were re-suspended in SDS sample buffer (60 mM Tris–HCl, 1% SDS, 10% glycerol, 0.05% bromophenol blue, pH 6.8, with 2% β-mercaptoethanol). Samples were subjected to 10% SDS-PAGE (Bio-Rad, Hercules, CA) and transferred to PVDF membranes (Bio-Rad). Transfer efficiency was determined by Ponceau S staining (Sigma-Aldrich). PVDF membranes were incubated with blocking solution (TBS containing 0.1% Tween 20 and 5% BSA) and were probed with specific antibodies (anti-PanH4ac (rabbit): # 39925 Active motif, Carlsbad, CA; anti-pan H4ac (mouse): 61337 #anti-PanH3ac: # 61638 Active motif; anti-H3 total: #61648 Active motif; anti-H4K12ac: # 39165 Active motif; anti-K5ac: # 39699 Active motif; anti-K8ac: # 2594S Cell Signaling, Danvers MA; anti-K16ac # 13534S Cell signaling; anti-GAPDH: Santa Cruz Biotechnology, Dallas, TX; anti-BRD4: Abcam Cambridge UK). Protein bands were detected using chemiluminescence kit (Millipore, Billerica, MA). ImageJ was used to quantify immunoblot results.