Profiling Breast Cancer Cell Lysates

The whole cell lysates from breast cancer cell lines (MCF10A, MCF7, T47D, MDA MB-231) were prepared using RIPA buffer (500 mM NaCl, 5 mM MgCl₂, 1% Na deoxycholate, 20 mM Tris-HCl (pH 8.0), 10% glycerol, 1 mM EDTA, 100 mM EGTA, 0.1% NP40, 1% Triton X-100, 0.1 M Na₃VO₄, 1X Protease inhibitor). Approximately 20–40 microgram of protein was separated using 10–12% SDS-polyacrylamide gel and transferred onto PVDF membrane (GE Healthcare Life Sciences, Chalfont, UK). Blots were incubated with 5% nonfat milk for blocking and were further incubated with 1 μg each of subsequent antibodies ERα (8644, Cell signaling technology, Danvers, MA, USA), ERRβ (Sc-68879, Santa Cruz) [37 (link)], α-tubulin (Sigma-Aldrich), cyclin D1 (2978, Cell Signaling Technology), p21^cip (2947, Cell Signaling Technology), p18 (2896, Cell Signaling Technology) followed by corresponding HRP labeled secondary antibody. The blot was incubated with ECL (Santa Cruz) for 5 min and visualized in Chemidoc XRS+ molecular 228 imager (Bio-Rad, Hercules, CA, USA). α-tubulin was considered as a loading control. The western blot images were quantified using Image J software (NIH, Bethesda, MD, USA).