2.5. Extraction of Protein and Western Blotting Analysis

Both of the cells and tissues were immediately washed using PBS and lysed in situ for 15 min with radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Pierce, Waltham, MA, USA) containing 100 µM Na₃VO₄, and 100× protease inhibitor cocktail (Thermo Fisher Pierce, Waltham, MA, USA). Following centrifugation for 15 min at 13,000 rpm, whole cell lysates were collected, and the protein concentration was determined using the Lowry method. Equal amounts of protein were then loaded onto 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to polyvinylidene fluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). PVDF membranes were incubated with bovine serum albumin (BSA) at 2%. (Sigma-Aldrich, St. Louis, MO, USA) in TBST (12.5 mM Tris/HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20) (Sigma-Aldrich, St. Louis, MO, USA) at 4 °C overnight. After washing with TBST three times, blots were incubated with primary antibodies diluted in TBST. After washing with TBST three more times, the blots were incubated with horseradish peroxidase (HRP) labeled secondary antibodies at room temperature for 1 h. Membranes were then rewashed thoroughly, and the binding results were detected using the enhanced chemiluminescence plus Western blotting detection system (Thermo Fisher Pierce, Waltham, MA, USA) in accordance with the manufacturer’s instructions. Finally, membranes were scanned and subjected to densitometry analysis (VisionWorks LS, UVP, CA, USA) in accordance with the manufacturer’s protocols. Specific antibodies that we used are listed in Supplementary Table S1.