Clarity western enhanced chemiluminescence substrate kit

HEK293 lysates were obtained using RIPA lysis buffer (Sigma Aldrich) with phosphatase and protease inhibitor cocktails (Roche). The protein concentrations in the samples were determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific). A total of 20 µg of protein samples diluted in sample buffer containing 2-mercaptoethanol (Sigma-Aldrich), denatured for 10 min at 95 °C, were loaded in each well and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in a 10 or15% polyacrylamide gel cast using the TGX FastCast acrylamide kit (Bio-Rad). After electrophoretic separations, proteins were transferred to polyvinylidene fluoride (PVDF) membrane using a transfer unit (Bio-Rad) at 80 V for 1 h. After transfer, the PVDF membranes were blocked with 5% bovine serum albumin (BSA) for 2 h and incubated overnight at 4 °C with primary antibodies. These include: anti-pSer209eIF4E (Cell Signaling Technology #9741), anti- eIF4E (Cell Signaling Technology, #9742, anti-4E-BP1(Cell Signaling Technology #9452) and anti-β-actin (Cell Signaling Technology #8H10D10). Detection of the immunoreaction was performed with a Clarity western enhanced chemiluminescence substrate kit (Bio-Rad) using ChemiDoc MP imaging system (Bio-Rad).