Western Blotting

The tissues were ground and lysed in a RIPA lysis buffer (Sigma-Aldrich) containing 1% protease inhibitor cocktail (Calbiochem) and 1% phosphatase inhibitor cocktail (P5726, Sigma-Aldrich). Following centrifugation, the supernatant was collected and protein content was evaluated by the Bradford assay or a BCA Protein Assay Kit (Thermo Fisher Scientific, cat # 23225). Twenty to 50 μg from each sample under reducing conditions was loaded into each lane and separated by electrophoresis on a 10% SDS polyacrylamide gel. Following electrophoresis, proteins were transferred to Cellulose Nitrate membranes (Tamar). Nonspecific binding was blocked by incubation with TBST [10 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.1% Tween 20] containing 5% skim milk for 1 hour at room temperature (RT). Membranes were subsequently incubated with antibodies (Western blot antibodies list; Supplementary Table S3).

Antibody was detected using peroxidase-conjugated AffiniPure goat anti-rabbit IgG or goat anti-mouse IgG (Jackson ImmunoResearch) and enhanced chemiluminescence Western blotting detection reagents (EZ-Gel, Biological Industries). Gels were quantified by Gel Doc XR+ (Bio-Rad) and analyzed by ImageLab 5.1 software (Bio-Rad). The relative intensity of each band was calculated by dividing the specific band intensity by the value obtained from the loading control.