Mouse anti β actin monoclonal antibody

Cells were grown and transfected with 25 nM of miRNA mimic or negative control RNA using DharmaFECT reagent. At 72 hours after transfection, cells were harvested and proteins extracted using radio immunoprecipitation assay lysis buffer (150 mM NaCl, 1% NP-40, 0.5% Na-DCA, 0.1% SDS, 50 mM Tris-HCl [pH 8.0], and protease inhibitor) and quantified by the Bradford assay method (Quick Start™ Bradford Protein Assay; Bio-Rad Laboratories Inc., Hercules, CA, USA). Thirty micrograms of protein was separated on 12% SDS-polyacrylamide gel and then transferred to nitrocellulose membrane (GE Healthcare Life Sciences Whatman™, Little Chalfont, Buckinghamshire, UK). The membrane was incubated with a mouse anti-TOMM20 monoclonal antibody (dilution, 1:5,000, Abcam, Cambridge, UK), or a mouse anti-β-actin monoclonal antibody (dilution, 1:5,000, ABM, Vancouver, BC, Canada,) overnight at 4°C and further incubated with a HRP-conjugated secondary antibody (goat anti-mouse 1:5,000, Santa Cruz Biotechnology Inc., Dallas, TX USA) for 1 hour at room temperature. Signals were detected using a enhanced chemiluminescence solution (Thermo Fisher Scientific). The β-actin gene (ACTB) was used as a loading control. The band intensity was analyzed using ImageJ software (version 1.49; National Institutes of Health, Bethesda, MD, USA) and defined as the ratio of target protein relative to β-actin gene.