Immunoblot analysis.
Whole cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.1% sodium deoxycholate) supplemented with 2 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 1:100 protease/phosphatase inhibitor cocktails (Sigma). Crude lysates were centrifuged at 13,000 rpm for 10 min at 4 °C and the supernatants were collected for further analysis. Protein concentrations were determined with the Bio-Rad DC protein assay system. Lysates were boiled in the presence of 2.5% β-mecaptoethanol, separated by denaturing SDS-PAGE, and transferred to 0.45-μm Optitran membranes (Schleicher & Schuell) in a Bio-Rad transfer unit. Membranes were immunoblotted with the appropriate primary antibody followed by horseradish peroxidase–tagged secondary antibodies (Amersham Biosciences and Dako) and detected with the SuperSignal West Pico System (Pierce).