Immunoblot Analysis

IPI-2I and PK-15 cells were rinsed twice with PBS and lysed with RIPA lysis buffer containing 20 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 10% glycerol, 0.1% SDS, and 0.5% sodium deoxycholate with protease inhibitor cocktail (1 mM Na₃VO₄, 20 μg/mL PMSF, 10 μg/μL leupeptin, and 50 mM NaF). Lysates were centrifuged at 13,000 rpm for 10 min. Concentrations of whole cell lysates were measured using Lowry assay (Bio-Rad, Hercules, CA, USA). Then 25 μg of total proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes using a Hoefer electrotransfer system (Amersham Biosciences, Amersham, UK). These transferred nitrocellulose membranes were incubated with anti-Ets1 and anti-β-actin antibodies (1:1000 dilution in 1% skim milk) at 4 °C overnight followed by incubation with a secondary antibody (1:5000 dilution in 1% skim milk) for 1 h at room temperature. Bands were visualized by enhanced chemiluminescence (Sigma-Aldrich) and analyzed using ChemiDoc (Davinchi-K, Seoul, Korea). Polyclonal rabbit anti Ets-1 and polyclonal mouse anti-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibodies were horseradish peroxidase conjugated anti-mouse IgG and anti-rabbit IgG (Thermo Scientific Pierce, Rockford, IL, USA).