SDS-PAGE and Western Blotting for Protein Analysis

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and western blotting were performed as described previously [29 (link)]. Cell lysates were prepared in lysis buffer [10 mM HEPES-KOH, pH 7.4, 100 mM KCl, 1 mM MgCl₂, 1% Triton X-100, and protease inhibitor cocktails (Nacalai)]. After incubation for 10 min on ice, the lysate was clarified by centrifugation at 14,000 × g for 10 min. The supernatants were then subjected to SDS–PAGE on a 7.5% gel (Bio-Rad). The gel was electrotransferred to polyvinylidene fluoride or polyvinylidene difluoride membranes (pore size: 0.45 μm, Merck Group Japan, Tokyo, Japan). The proteins on the membrane were detected by incubation with diluted anti-AhR, anti-ARNT (Santa Cruz Biotechnology, Cosmo Bio Co., LTD, Tokyo, Japan), and anti-γ-tubulin (Sigma, Sigma-Aldrich Japan, Tokyo, Japan) followed by HRP-conjugated anti-mouse immunoglobulin G (Cell Signaling, CST Japan, Tokyo, Japan) using a charge-coupled device camera.