Analyzing Protein Expression in HEK-293T Cells

HEK-293T cells were transfected with the gene of interest for either 24 h or 36 h. The cells were harvested and lysed in radioimmunoprecipitation assay lysis buffer (1% NP-40, 20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% sodium deoxycholate, and 1 mM Na₃VO₄). Protein estimation was carried out by using BCA Protein Assay Kit (Pierce, Thermo Scientific). An equal amount of protein was loaded on SDS-PAGE and transferred to nitrocellulose membrane as described before (24 (link)). The membranes were blocked with 5% nonfat dry milk (Himedia Laboratories) or 5% bovine serum albumin. The primary antibodies used were anti-AKT, anti-GAPDH, anti-phospho-AKT (S-473) (Cell Signaling Technology), anti-Myc, anti-HA (Clontech), ab140601 (abcam–K48 antibody), and anti-GST (Santa Cruz Biotechnology). The secondary antibodies used were anti-rabbit/mouse-horseradish peroxidase conjugated (Jackson ImmunoResearch). Blots were developed using Enhanced Chemiluminescence) reagent.