Primary antibody for er α

MCF-7-vector and MCF-7-miR-335 cells were grown in 10% FBS DMEM. Cells were washed with phosphate-buffered saline (PBS) and lysed with M-Per lysis buffer supplemented with 1% protease inhibitor and 1% phosphatase inhibitors (I/II) (Invitrogen). Supernatant containing protein extracts was obtained through centrifugation at 12,000 rpm (5415, Eppendorf, Westbury, NY, USA) for 10 min at 4 °C. Protein extracted per sample was determined by absorbance at 260 and 280 nm. Proteins were heat denatured and loaded on Bis-Tris-nuPAGE gel (Invitrogen). Protein transfer to nitrocellulose through iBlot and iBlot transfer stacks was per the manufacturer’s protocol (Invitrogen). Nonspecific binding was blocked by incubation in 3% milk (in 1% Tris buffered saline-Tween (TBS-T)) for 1 h. Overnight incubation of membrane with primary antibody for ERα (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:400 at 4 °C followed by 3 × 15 min washes in 1% TBS-T. Membranes were incubated for 1 h in secondary antibody 1:10,000 dilution (LiCor Bioscience, Lincoln, NE, USA) followed by 3 × 10 min washes in 1% TBS-T. Band density was determined by LiCor gel imager. Normalization was to Rho GDI-α (Santa Cruz Biotechnology).