I8896

HeLa and NSC-34 cells were seeded at a density of 2 × 10⁵ cells/well in 12-well plates. Twenty-four h later cells were treated with varying concentrations of BafA1 (0, 1, 10 and 20 nM). Sixteen h after treatment the media was removed from the cells, the cells were washed in PBS and then harvested in fresh PBS. Cells were then pelleted at 380 g for 5 min in a table-top centrifuge and the PBS was removed. Cells were lysed in 300 μL of RIPA buffer (150 mM NaCl, 1% IGEPAL® CA-630 [Sigma-Aldrich®, I8896], 0.5% sodium deoxycholate [Sigma-Aldrich®,D6750], 0.1% SDS [Fisher Scientific, BP166], 50 mM Tris, pH 8.0, including protease [Sigma-Aldrich, P8340] and phosphatase [Sigma-Aldrich, P5726] inhibitors [at a 1:1000 dilution]). A BCA assay (ThermoFisher Scientific, 23225) determined the total protein concentration of each lysate and then lysates were separated by SDS PAGE on a 5–20% acrylamide gels with 20 μg of total protein loaded per well. Gels were transferred overnight and autophagy marker proteins were revealed by western blotting with the mouse anti-SQSTM1 lck ligand antibody, rabbit anti-SQSTM1 antibody (Enzo Life Sciences, BML-PW9860) and rabbit anti-LC3B antibody (MBL International, PM036). The blots were also stained for ACTB/β-actin (Sigma-Aldrich, A1978) to confirm equal protein loading.