Protein Extraction and Western Blot Analysis

Cell extracts were prepared in lysis buffer containing 20 mM Tris HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5% sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 µg/µL leupeptin, protease inhibitors (#P8340; Sigma-Aldrich) and phosphatase inhibitors (#P5726; Sigma-Aldrich). Protein concentrations were determined using the BCA reagent as described earlier, and samples were denatured using SDS sample buffer (#1610747; BioRad). Samples were loaded into a Criterion Tris-Glycine Extended Gel (#5671124; BioRad) and separated by electrophoreses at 60 mA. The gels were then transferred onto a nitrocellulose membrane (#1620115; BioRad) by a wet transfer system (BioRad) at 100V for 1 h at room temperature. All membranes were then blocked by incubation with 5% dry milk in TBST (TBS with 0.1% Tween20) for 1 h at room temperature. Membranes were probed with the primary antibody overnight at 4°C in the blocking buffer, washed with TBST, and incubated with the peroxidase-conjugated secondary antibody. Enhanced chemiluminescence (ECL) Western blotting substrates (#170-5061; BioRad) were used for the visualization of the results. The acquisition of images was performed using the ChemiDoc MP Imaging System (BioRad).