Whole-cell Protein Extraction and Western Analysis

Cell pellets were resuspended using lysis buffer (25 mM HEPES, pH 7.4, 0.4 M KCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 1% NP-40, 1 mM PMSF, and 2 μg/mL of leupeptin) of 2.5× volume of cell pellets. After three cycles of freeze/thaw, lysates were kept on ice for 30 min and were then centrifuged at 16,000 g for 10 min at 4 °C. The supernatants were defined as whole-cell lysates and were subjected to BCA assay and LI-COR Western analysis. The protocol for Western analysis was described previously [48 (link)] with minor modification. Fifteen percent acrylamide gels were transferred for 3 h at 4 °C. After the wet transfer, Total Protein Staining was performed using LI-COR Total Protein Stain. Membranes for the examination of LC3B levels for autophagic flux were dried overnight and wet with PBS before blocking. The transferred nitrocellulose membranes were blocked in PBS with 5% BSA for 1 h. Dilutions for antibodies were as follows: 1:1,000 for p23 (JJ3), p62, LC3B and AHR (A-3x); 1:2,000 for AHR (SA210); 1:5,000 for β-actin; 1:200 for HSP90 (N-17). If not specified, Western bands were normalized using Total Protein Stain. Results were obtained and analyzed using an LI-COR Odyssey CLx imaging system.