Hdac3 antibody

Cultured NRVM were infected with GFP-CapZβ1 adenovirus. After 24 h of infection, cells were washed twice in ice-cold PBS and lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP40, 0.1% SDS) plus phosphatase inhibitors (Sigma Aldrich, #P5726, P0044) for 1 h at 4 °C under constant agitation. Following protein extraction, protein lysates were precleared using 25 μL Protein A/G Plus-Agarose beads (Santa Cruz, #sc-2003) for 1 h at 4 °C. Precleared lysates were incubated 24 h with 2 μg of HDAC3 antibody (Cell Signaling Technology; 4668) at 4 °C, then immunocomplexes were isolated by adding Protein A/G Plus-Agarose beads overnight at 4 °C. Beads were washed three times in the binding buffer. SDS-PAGE and Western blotting were performed using 12% Mini-PROTEAN® TGX^™ Gel (Bio-Rad Laboratories, #456-1044). Polyvinylidene difluoride (PVDF) membranes were incubated with GFP primary antibody (Enzo Life Sciences, ADI-SAB-500), followed by horseradish peroxidase (HRP) secondary antibody (anti-mouse) for 1 h at room temperature. Proteins were finally treated with an ECL Plus kit and visualized with the aid of ChemiDoc XRS+ and analyzed with Image Lab (Bio-Rad Laboratories).