Cells were lysed in radioimmunoprecipitation assay buffer [50 mM tris (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS]. Protein content was then tested using the bicinchoninic acid assay (Pierce), and equal amounts of lysates were boiled in Laemmli buffer for 10 min at 95°C. Samples were then subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE). After blotting onto Hybond P polyvinylidene fluoride membranes (GE Healthcare), blots were blocked with 5% milk in tris-buffered saline with Tween 20 (TBS-T), and primary antibody, in 5% BSA in TBS-T, was added at 4°C overnight. The protein was then detected by using an HRP-conjugated secondary antibody and visualized with LumiGLO (Cell Signaling Technology).

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