Anti hdac2 antibody

For SDS-PAGE, similar amounts of control and test group SK-N-MC cellular protein, typically 40 μg per lane were used. All the protein samples were separated by using Any KD Mini-Protean TGX precast Gels (Bio-Rad, Cat # 456–9034). Proteins were transferred to nitrocellulose membranes, and the quality of protein measurement, electrophoresis, and transfer was checked by staining with Ponceau S. Membranes were blocked with 5% skimmed milk in TBS-T (20 mM Tris buffer, pH 7.5, 0.5 M NaCl, 0.1% Tween 20) for 1 hr at room temperature and incubated at 4°C overnight in the primary antibody diluted in 2% skimmed milk in TBS-T. The primary antibodies used were as follows: anti-HDAC2 antibody (Millipore cat # 07–222, 1/5000); rabbit anti-phospho-eIF2α (Ser51) antibody (Cat # 9721, 1/1000), rabbit anti-eIF2α antibody (Cat # 9722, 1/1000) from Cell Signaling (Beverly, MA). Subsequently, blots were washed in TBS-T (4 times, 10 min each) and incubated for 1 hr at room temperature in horse radish peroxidase-goat anti-rabbit antibody (Promega, Cat # W401B) diluted 1/2500 in 2% skim milk in TBS-T. After additional washings, protein bands were detected by chemiluminiscence using SuperSignal West Pico Luminol/Enhancer (Thermo Scientific, Cat # 1856136) and SuperSignal West Pico substrate (Thermo Scientific, Cat # 1856135). Band density was measured by using ImageJ software.

In the SK-N-MC cells infected with HIV and/or treated with nicotine/vorinostat combination (as explained above), expression levels of HDAC2 was measured by qPCR with the use of Taqman assay (Assay ID # Hs00231032_m1) and by western blot using anti-HDAC2 antibody (Millipore cat # 07–222).