Estrogen receptor α

Cell lysates prepared using Laemmli sample buffer(2% SDS, 10% glycerol, 62.5 mM Tris-HCl, pH 6.8 and 50mM dithiothreitol , DTT). The protein content measured by BCA Protein Assay Reagent (Thermo Sientific, US). Lysates were denatured by heating to 95°C for ten minutes and 50µg of each sample loaded in each lane on a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS -PAGE) and subjected to gel electrophoresis. The proteins were transferred to PVDF membrane (Roche, Germany) and blots were blocked overnight at 4°C in casein blocking buffer (10% casein in TBS and 0.1% tween 20). Blots were then incubated at 4°C, overnight with primary antibodies to p53, DYKDDDDK Flag, Estrogen Receptor α, Bcl2, Bax (all from Cell signaling, MA, USA) and β-Actin (Santa Cruz, CA, USA). Following primary antibody incubation, the blots were washed and incubated with goat anti mouse/rabbit horse radish peroxidase conjugate (Biorad, Richmond, CA, USA) for 60 minutes at room temperature and developed with the BM chemiluminescence system as directed by the manufacturer (Roche, Germany). In all western blotting experiments, β-Actin was used as a loading and internal control. The quantitation of the protein bands intensity was done by ImageJ software (NIH, MD, USA).