Polyclonal anti c myc antibody

Cells were lysed in a lysis buffer (10 mM KCl, 20 mM HEPES, 5 mM EDTA, 1% NP-40, 0.25% deoxycholate, pH 7.4) with protease and phosphatase inhibitors (1 mM Na3VO4, 10 mM NaF, 1 mM phenylmethanesulfonyl fluoride, 2 μg/ml aprotinin). Protein concentrations were measured by the BCA protein assay (Pierce, IL, USA). Equal amounts of the protein were denatured in sample buffer and then electrophoresed by 5-10% SDS-PAGE, transferred to the polyvinylidene fluoride (PVDF) membrane (no. IPFL00010; Merck Millipore, Darmstadt, Germany) under 100 V for 2 h and incubated with the following primary antibodies overnight at 4°C: anti-β-actin antibody (Cell Signaling Technology); Mouse monoclonal anti-E-cadherin (#14472, Cell Signaling Technology), rabbit polyclonal anti-β-catenin (ab6302, Abcam); mouse monoclonal anti-cycle D1 antibody(MA1-12296, Thermo Fisher Scientific); polyclonal anti-c-myc antibody (#9402, Cell Signaling Technology). The primary antibody incubation was followed by incubation with fluorescent Dye-tagged secondary antibodies that were 20,000-fold diluted in Odyssey blocking buffer (TBS) and an Odyssey Infrared Imaging System (LI-COR).