Protein Extraction and Western Blot Analysis

Proteins were extracted from the treated cells using cell lysis buffer (Cell Signaling Technology) containing a protease-inhibitor mixture (Thermo Scientific, Rockford, IL, USA) and phosphatase-inhibitor mixture (Nacalai Tesque, Kyoto, Japan). In some experiments, the nuclear and cytoplasmic fractions were extracted using NE-PER (Thermo Fisher Scientific) according to the manufacturer’s instructions. Protein concentrations were measured using a DC protein assay kit (Bio-Rad, Hercules, CA, USA). Equal volumes of protein sample were loaded on precast 12.5% polyacrylamide gel (SuperSepTM Ace; Fujifilm Wako Pure Chemical Co) and electrophoresed in Tris–glycine sodium dodecyl sulfate (SDS) running buffer. The subsequent analyses (blotting, antibody reaction and band detection) were conducted following previously reported protocols [47 (link)] with primary antibodies against Iκ-Bα, p65, phospho-JNK, JNK, phospho-p38 MAPK, p38 MAPK, phospho-ERK, ERK, phopho c-Jun, c-Jun and β-actin. The band intensity of each blot was quantified by densitometric analysis using Image LabTM^® 2.0 software (Bio-Rad).