β actin antibody

After transfection with miR-21 mimic or inhibitor for 48 h, cells were harvested and lysed using RIPA buffer (Cell Signaling Technology, Dancers, MA, USA) supplemented with phenylmethylsulphonyl uoride (Nacalai Tesque, Kyoto, Japan). Equal amounts of protein were runned by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene uoride membranes (0.45 µm; Millipore). Membranes were washed with Tris-buffered saline (TBST) containing 0.1% Tween-20 (Nacalai Tesque) and 5% bovine serum albumin (Sigma-Aldrich, Saint Louis, MO, USA), and incubated overnight at 4°C with primary antibodies (PDCD4 antibody from Cell Signaling Technology, Danvers, MA, USA; β-actin antibody from MP Biomedicals, LLC, Irvine, CA, USA). After washing with TBST, membranes were further incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000; Santa Cruz Biotechnology, Dallas TX, USA) for 1 h at room temperature, and developed by using an electrochemiluminescence system in the end (GE Healthcare, Little Chalfont, UK). Protein bands were detected by an LAS4000mini imaging system (Fuji lm, Tokyo, Japan), and band intensities of Western blots were quantitatively measured by calculating integrated grayscale densities in consistently sized windows incorporating each band using ImageJ version 1.48 software as described previously [21] .