Protein Extraction and Western Blotting

Protein lysates were obtained by collecting cells in a lysis buffer (20 mM HEPES, 250 mM NaCl, 2 mM EDTA, 1% SDS, 10% glycerol, 50 mM NaF, 0.1 mM hemin chloride, 5 mM NEM, 1 mM PMSF and 10 mg/mL leupeptin and aproptinin) [6] (link), [47] (link), [48] (link) followed by sonication. Protein concentration was performed using the BCA assay (Thermo Scientific). Equal amounts of protein extract (9–15 µg depending on experiment) from each lysate were resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane as described. The blots were blocked overnight with 5% nonfat dry milk and incubated with specific antibodies. The antibodies used are detailed in Table 2. After incubation with the appropriate secondary antibody, signals were detected using immunochemiluminescent reagents (GE Healthcare, Piscataway, NJ). Equal protein loading in each lane was confirmed with a β-actin antibody (#A300-491, Bethyl).