Anti p ask1

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.

As previously described [31 (link)], cell lysates were diluted in Laemmli buffer, separated by polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to PVDF membranes. Membranes were blocked in 5% non-fat dry milk before incubating overnight at 4˚C in rabbit polyclonal anti-Trx2 (1:250, Santa Cruz, sc-50336), rabbit polyclonal anti-Prx3 (1:1,000, Thermo Fisher Scientific, LF-PA0030), rabbit polyclonal anti-TrxR2 (1:1,000, Abcam, ab58445), rabbit polyclonal anti-ASK1 (1:500, Santa Cruz, sc-7931), rabbit polyclonal anti-pASK1 (1:1,000, Cell Signaling, 3765), rabbit polyclonal anti-Bax (1:1,000, Santa Cruz, sc-493), mouse monoclonal anti-Bak (1:1,000, EMD Millipore, AM03) or rabbit polyclonal anti-β-actin (1:1,000, Sigma Aldrich, A2066). Membranes were then incubated with anti-rabbit or anti-mouse (1:5,000, Southern Biotechnology) HRP-conjugated secondary antibodies and immune complexes were detected by chemiluminescence captured and analyzed on a UVP bioimaging system (Upland, CA). Semi-quantitative analyses of immunoblots were performed using Image Studio (LI-COR).