Rabbit anti eif2α antibody

Protein was extracted from EndoC-βH1 cells in radioimmunoprecipitation assay buffer (Sigma-Aldrich) supplemented with halt protease and phosphatase inhibitor Cocktail (Thermo Fisher Scientific). Protein samples were loaded onto NuPAGE 4–12% Bis–Tris Protein Gels (Thermo Fisher Scientific), resolved by electrophoresis and transferred onto nitrocellulose membranes. Membranes were incubated with the following primary antibodies: mouse monoclonal anti-β-actin antibody (Invitrogen, MA1-140; 1:20,000), rabbit monoclonal anti-phospho CHEK2 T68 antibody (Cell Signaling, 2197S; 1:1,000), mouse monoclonal anti-CHEK2 antibody (Cell Signaling, 3440T; 1:1,000), rabbit anti-eIF2α antibody (Cell Signaling, 5324; 1:1,000), rabbit anti-phospho eIF2α Ser51 antibody (Cell Signaling, 3597; 1:1,000), rabbit anti-α-tubulin antibody (Cell Signaling, 2144; 1:1,000), rabbit anti-phospho PLK1 T210 (Abcam, ab155095; 1:200) and mouse anti-PLK1 (Abcam, ab17056; 1:1,000). Primary antibodies were detected by fluorophore-conjugated secondary goat anti-rabbit (LI-COR IRDye 800CW, 926-32213; 1:15,000) and donkey anti-mouse (LI-COR IRDye 680RD, 926-32210; 1:15,000) using LI-COR Odyssey Imagers. Western blot images were analyzed with Image Studio software 5.2.5.

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.