Mouse anti rb

Samples were harvested in phosphate-buffered RIPA lysis buffer (Boston BioProducts, Ashland, MA). After incubation on ice for 30 min, samples were centrifuged at 12,000 rpm for 10 min and the supernatants were collected. Total protein concentrations were determined by the Pierce BCA protein Assay (Thermo Scientific, Rockford, IL). Protein lysates were boiled and separated on 10% SDS-PAGE gels and transferred to PVDF membranes. Following blocking, membranes were incubated with primary antibodies (diluted accordingly in 5% BAS/TBST). Primary antibodies and dilutions used were rabbit anti-ZBTB46 (Novus, NBP1-88506, 1:500), mouse anti-Rb (Cell Signaling 9309, 1:1000), rabbit anti-phospho(Ser797)-Rb (Cell Signaling 9301, 1:1000), mouse anti-β-actin (Sigma-Aldrich, A5316, 1:10,000), and rabbit anti-GAPDH (Santa Cruz, sc-25778, 1:10,000). After overnight incubation with the primary antibody at 4 °C, membranes were washed and incubated with secondary antibodies (anti-mouse IgG-HRP (Santa Cruz, sc-2005, 1:10,000), anti-rabbit IgG-HRP (Santa Cruz, sc-2004, 1:10,000)), diluted in 5% BSA/TBST for 1 h at RT. Bands were developed by adding Immobilon Western chemiluminescent HRP substrate (Millipore, Billerica, MA) to the membranes and captured using Kodak image station 4000 mm Pro. Protein content was quantified using ImageJ and normalized to GAPDH or β-actin.