Immunoblotting Analysis of Autophagy Markers

Protein sample preparation and Western immunoblotting were performed as previously described (Qin et al., 2011 (link); Pandey et al., 2017 (link)). Primary antibodies used in the immunoblotting analysis included anti-p-ULK1, anti-ULK1, anti-ASK1, anti-p-ASK1, anti-SAPK/JNK, anti-p-SAPK/JNK (Cell signaling); anti-AMPKα, anti-p-AMPKα, anti-Atg9a (Thermo Scientific); anti-LC3, anti-p-ASK1 and anti-GAPDH (Santa Cruz Biotech., Inc); anti-BAK, anti-BAX (EMD Millipore), anti-p-IRE1α (GeneTex, Inc), anti-IRE1α (Novus Biologicals). Dilution of primary antibodies was 1:1,000. Secondary antibody HRP anti-IgG (Sigma-Aldrich, USA, 1:1,000~5,000) was used in the immunoblotting analysis. Densitometry of blots was performed using the ImageJ software package (http://rsbweb.nih.gov/ij/). The relative expression levels of target proteins and the ratio of blot LC3-II/LC3-I at the indicated time points were calculated as previously described (Qin et al., 2011 (link)). All Westerns were performed in triplicate and representative images are shown.