Rabbit antihuman gapdh antibody

Whole protein lysate was isolated using RIPA buffer [150 mMNaCl, 50 mMTris-HCl (pH = 8), 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS] completed with 100X protease/phosphatase inhibitor cocktail (Thermo Fisher, Massachusetts, USA), and protein concentration was measured by BCA protein assay kit (Thermo Fisher). Each lysate from different samples was separated by using Mini-PROTEAN TGX 10% precast gel (Bio-Rad, California, USA) and transferred to nitrocellulose membrane using Trans-Blot Turbo transfer system (Bio-Rad). Membranes were blocked with 5% BSA (Sigma–Aldrich) in TBS-T and then incubated with a mouse antihuman TUBB1 antibody (OriGene, TA506654; 1:3000 dilution) at 4 °C overnight, an anti-Flag HRP antibody (Abcam, ab49763; 1:1000 dilution, Cambridge, UK) at room temperature for 1 h, a rabbit antihuman GAPDH antibody (Cell Signaling, 5174; 3:10000 dilution) at room temperature for 1 h, or a rabbit antihuman β-actin antibody (Cell Signaling, 8457; 1:1000 dilution) at room temperature for 1 h. Upon washing with TBS-T, membranes were incubated with compatible HRP-conjugated antimouse IgG (Cell Signaling, 7076; 1:5000 dilution) or antirabbit IgG (Cell Signaling, 7074; 1:5000 dilution) secondary antibodies at room temperature for 1 h. Protein bands were detected using ECL Plus Western blotting substrate (Thermo Fisher) and ChemiDoc XRS+ imaging system (Bio-Rad).

Rabbit anti-human phospho-Akt (pAkt Ser-473) antibody, rabbit anti-human pan-Akt antibody, rabbit anti-human caspase-3 antibody, rabbit anti-human caspase-7 antibody, rabbit anti-human cleaved caspase-3 antibody, rabbit anti-human cleaved caspase-7 antibody, and rabbit anti-human GAPDH antibody were purchased from Cell Signaling Technology (Danvers, MA). Rabbit anti-human PARP antibody was purchased from Abcam (Cambridge, MA). Goat anti-rabbit IgG -HRP conjugated was purchased from Thermo Scientific (Waltham, MA). The PI3K inhibitor, LY294002, was purchased from Cell Signaling Technology (Danvers, MA). Sorbitol was purchased from US Biological Corporation (Boston, MA). Cyclohexamide (CHX), a eukaryotic protein biosynthesis inhibitor, purchased from Sigma-Aldrich Corp (St. Louis, Missouri).