Reblot

Subconfluent CRL-11515 cells treated with 0.001% or 0.004% BAK for 1 minute in the absence or presence of lacritin were lysed (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 0.5% SDS, and 1:100 protease inhibitor) to extract protein. Total protein was measured by the Micro BCA Protein Assay kit (Thermo Scientific, Nepean, ON) and 20 μg of protein was loaded per well. Protein was separated on a 10% PAGE gel and transferred to a nitrocellulose membrane. Autophagy was estimated by Western blotting for the autophagy marker LC3-II using anti-LC3-II antibody (Sigma-Aldrich, St. Louis, MO) at a dilution of 1:1,000 followed by anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:3,000) and signal was detected by enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL) with the ChemiGenius 2 Imaging System (Syngene, Frederick, MD). To ensure equal protein loading, nitrocellulose membranes were stripped (REblot; Chemicon International, Temecula, CA) and reprobed with anti-GAPDH antibody at a dilution of 1:5,000 (Cedarlane Laboratories, Hornby, ON, Canada). Intensities were quantified using the FluorChem 8000 (Alpha Innotech, San Leandro, CA) and corrected for background. The densitometric ratio of lipidated LC3-II/GAPDH is presented.