Western Blot Protocol for Protein Analysis

Cells were lysed in ice-cold RIPA buffer supplemented with Pierce™ protease and phosphatase inhibitor mini tablets (Thermo Scientific). Protein concentration was measured using the Pierce™ Rapid Gold BCA Protein Assay Kit and 30–40 µg protein samples were run on 10% SDS gels for protein separation, followed by blotting the gels on 0.2 µm nitrocellulose blotting membrane (Amersham, Freiberg, Germany) at 300 mA for 1 h in a cold room. After blotting, membranes were blocked with 5% skimmed milk for 1 h. Antibody concentrations used were as follows: β-actin, 1:10,000 (NB600-532SS, Novus Biologicals); LC3, 0.2 µg/mL (L8918, Merck) [31 (link)].