Goat anti rabbit igg 31460

After treatments, cells were lysed in ice-cold lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Na₄P₂O₇, 1 mM β-glycerophosphate, 1 mM Na₃VO₄) supplemented with protease (Fisher Scientific, Pittsburgh, PA, #P1-88266) and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, #04906845001), and Western blot was performed as previously described [44 (link)]. Membranes were incubated with primary antibodies pRb T821/826 #271930 (1:200), Rb #73598 (1:200), Cleaved Caspase 3 #9661 (1:1,000), Cleaved PARP #5625 (1:1,000), c-Myc #5605 (1:1,000), MCL-1 #4572 (1:1,000) (Cell Signaling Technology, Danvers, MA), VHL #564183 (1:500) (BD Biosciences, San Jose, CA), HIF-2α #NB100122 (1:1,000, Novus Biologicals, Littleton, CO), and β-actin #A5441 (1:5,000, Sigma Aldrich, St. Louis, MO) in 5% BSA/PBS/0.05%Tween20 (PBS-T) overnight at 4°C, and secondary antibody incubation with Goat anti-Rabbit IgG #31460 (1:5,000) (Thermo Fisher Scientific, Waltham, MA) and Goat anti-Mouse IgG #31430 (1:2,000) (Thermo Fisher Scientific, Waltham, MA) in 5% nonfat milk/PBS-T blocking buffer for 1 hour at room temperature (RT). The HRP signal was developed using ECL WB substrate (GE Healthcare, Scottsdale, AZ, USA; #RPN2236). Images were acquired on the Bio-Rad ChemiDoc XRS⁺ imaging system (BioRad, Hercules, CA).