Immunoprecipitation and Immunoblotting Protocol

Tissues were lysed in lysis buffer A [50 mM Hepes-NaOH (pH 7.5), 3 mM MgCl₂, 150 mM NaCl, 1 mM dithiothreitol, 1 mM phenylmethane sulfonylfluoride, leupeptin (1 μg/ml), 1 mM EDTA, 1 mM Na₃VO₄, and 10 mM NaF] containing biochemical detergents (0.5% NP-40, 1% CHAPS, and 0.03% SDS). Unless otherwise indicated, all lysis steps were performed at 4°C (12 (link), 36 (link)). For immunoprecipitations, the supernatants cleared by centrifugation were mixed with an antibody-absorbed protein G resin (GE Healthcare) or ExtraCruz (Santa Cruz Biotechnology) according to the respective manufacturer’s instructions. The immunoprecipitates or proteins in the cell supernatants were denatured, subjected to SDS–polyacrylamide gel electrophoresis, and blotted to polyvinylidene difluoride membranes using the TransBlot TurboTransfer System (Bio-Rad). The membranes were blocked with Blocking One (Nacalai) and immunoblotted using a primary antibody followed by a peroxidase-conjugated secondary antibody. The bound antibodies were detected using Chemiluminescence One (standard and strong detection reagents; Nacalai). The bands were scanned using a C-DiGit Blot Scanner (MS Systems). They were densitometrically analyzed to identify their quantification using UN-SCAN-IT software (Silk Scientific).