Imagequant digital imager

Cells were incubated in lysis buffer (Cell Signaling Technology) containing protease inhibitor mixtures (Roche Molecular Biochemicals, Indianapolis, IN) and phenylmethylsulfonyl fluoride (PMSF) at 4 °C for 30 min. The protein concentration was determined using bicinchoninic acid (BCA) assay (Pierce Biotechnology, Rockford, IL, USA). Proteins (30 − 60 µg) were separated by 12% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST for 1 h at room temperature and incubated with appropriate primary antibodies overnight at 4 °C, followed by secondary antibodies for 1 h at room temperature. Immunoblots were detected by Immunobilon Western Chemiluminescent HRP substrate (Millipore, Burlington, MA) and visualized on an ImageQuant^TM digital imager (GE Healthcare, Pittsburgh, PA).

Cells were incubated in lysis buffer (Cell Signaling Technology) and protease inhibitor mixtures (Roche Molecular Biochemicals, Indianapolis, IN, USA) at 4 °C for 30 min. The protein concentration was determined using bicinchoninic acid (BCA) assay (Pierce Biotechnology, Rockford, IL, USA). Proteins (30–50 µg) were separated by 12% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST for 1 h at room temperature. Subsequently, membranes were incubated with appropriate primary antibodies overnight at 4 °C and incubated with secondary antibodies for 1 h at room temperature. Immunoblots were detected by enhanced the chemiluminescence system and visualized on an ImageQuant^TM digital imager (GE Healthcare, Pittsburgh, PA, USA).