Western Blotting Experimental Workflow

Western blotting was performed as described (21 (link), 22 (link)). Cultured cells were washed with ice-cold PBS and lysed with RIPA lysis buffer (R0278; Sigma-Aldrich) containing protease and phosphatase inhibitor cocktails (P8340, P5726, and P0044; Sigma-Aldrich). Protein concentrations were determined using a BCA Protein Assay Kit (23227, Thermo Fisher Scientific). All samples were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. For blocking, 5% skim milk in Tris-buffered saline with 0.1% Tween-20 (TBS-T) or 5% bovine serum albumin (BSA) in TBS-T was used for non-phosphorylated and phosphorylated proteins, respectively. After blocking, the membranes were incubated with the indicated primary antibodies, followed by incubation with appropriate horseradish peroxidase-conjugated species-specific secondary antibodies. Bands were detected using the SuperSignal West chemiluminescent substrate (Thermo Fisher Scientific) and visualized using ImageQuant LAS-4000 (GE Healthcare, Little Chalfont, UK). Actin was used as the loading control to normalize the amount of protein.