Lumiglo peroxidase chemiluminescent substrate kit

Cells of interest were harvested, and protein extracts were obtained after cell lysis in RIPA buffer supplemented with a cocktail of protease inhibitors. Protein concentrations were determined using the Thermo Fisher BCA assay (Pierce). Equal protein amounts were run on Bolt 4%–12% Bis-Tris Plus gels (Invitrogen) and transferred to polyvinylidene membranes by electroblotting. Membranes were blocked with 5% nonfat dried milk/TBST buffer (Blotto) for an hour, incubated for an hour with primary antibody, washed with TBS and 0.05% Tween (TBST), and incubated again with secondary antibody in Blotto solution. Detection was performed via the LumiGLO Peroxidase Chemiluminescent Substrate Kit (Cell Signaling). The same membranes were re-probed after stripping to control for equal loading using β-actin as a control. Antibodies: E2F1: anti-hE2F1 3742S (1:1000, Cell Signaling); Rb: anti-Rb clone G3–245 (1:250, BD Biosciences); β-actin: anti-β-actin 8H10D10 (1:1000, Cell Signaling); Secondary antibodies: anti-Rabbit or anti-Mouse Ig-conjugated with HRP (1:1000, Cell Signaling). When indicated, E2^vF1 protein level were quantified and normalized against β-actin levels using the ImageJ gel module.