Chemidoc xrs molecular 228 imager

Western blots were performed using whole-cell lysates extracted using radioimmunoprecipitation assay buffer and as described previously.^{68 (link)} Nuclear and cytoplasmic extracts were fractionated using a CelLytic NuCLEAR Extraction Kit (Sigma) following manufacturer's instructions. Antibodies used include α-tubulin (T5168, Sigma), ERα (8438), cyclin D1 (2978), cyclin D3 (2936), p21^Cip (2947), HDAC1 (5356), HDAC2 (5113), HDAC3 (3949), HDAC4 (7628), HDAC6 (7558), acetylated histone 3 (9649), p18 (2896) and cleaved CASP7 (8438, CST), CASP7 (ab32522, Abcam), H3 (sc-8654), p300 (sc-584) and pol II (sc-899, Santa Cruz) and p53 (550832, BD Biosciences, East Rutherford, NJ, USA). Cleaved CASP7 (8438) detects both cleaved and uncleaved CASP7. Images were acquired by using Chemidoc XRS+ molecular 228 imager (Bio-Rad, Hercules, CA, USA). Western blot images were quantified using Image J software (NIH, Bethesda, MD, USA).

The whole cell lysates from breast cancer cell lines (MCF 10A, MCF-7, ZR-75-1, T-47D, SKBR3, MDA-MB-231 and MDA-MB-468) were obtained by 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, 1X Phosphatase Inhibitor). Protein concentration was calculated by the Bradford protein assay method. Total protein extracts (40 μg) were electrophoresed in 10% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membrane (GE Healthcare Life Sciences, Chalfont, UK). Blots were incubated with 5% skimmed milk (MP Biomedicals, India) for 1 h of blocking. Then the membrane was cut prior to incubation with primary antibodies and kept at 4°C overnight with gentle shaking (details of all antibodies and reagents are provided in the (Supplementary Table 1)). The membrane was then washed with 1X TBS-T and incubated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibody (1:5000 dilution) for 1 h. After washing, the blots were developed using Western Blot Chemiluminescence HRP Substrate (Takara Bio Inc.) in Chemidoc XRS+ molecular 228 imager (Bio-Rad, Hercules, CA, USA). The images were quantified using Image J software (NIH, Bethesda, MD, USA).

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).