Tissue preparation for Western blot analysis

Mice were perfused with 1× PBS, and tissues were dissected and snap‐frozen with liquid nitrogen and kept at −80°C. On the day of the experiment, frozen tissues were thawed and homogenized on ice with bead homogenizer (Moni International) in ice‐cold RIPA buffer (150 mM NaCl, 50 mM Tris–HCl [pH 8.0], 1% Triton X‐100, 0.5% sodium deoxycholate, 0.1% SDS) with 1 mM PMSF, proteinase, and phosphatase inhibitors. After centrifugation at 14,000 g for 15 min at 4°C, supernatants were collected as the RIPA‐soluble fraction. The insoluble pellets were washed with RIPA buffer and extracted in 2× v/w of Urea buffer (7 M Urea, 2 M Thiourea, 4% CHAPS, 30 mM Tris, pH 8.5). After sonication, samples were centrifuged at 200,000 g at 24°C for 1 h and the supernatant collected as the Urea‐soluble fraction. Protein concentrations were determined via BCA assay and then standardized. Equal amounts of protein were analyzed by Western blotting using the indicated antibodies.