Ecl chemiluminescent detection reagents

Western-blotting analysis was performed as previously described (Liang et al., 2019 (link)). Briefly, 40 µg of the total cell lysates were electrophoresed on a 4%–15% gradient SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel (BioRad, Hercules, CA), and the proteins were subsequently transferred onto a PVDF membrane (EMD Millipore, Billerica, MA). The membrane was sequentially immunoblotted with rabbit anti-XBP1 polyclonal antibody that recognizes both XBP1U and XBP1S (1:4000, Abcam, Cambridge, MA), horseradish peroxidase (HRP)-conjugated rabbit polyclonal anti-phosphorylated IRE1α (pSer724) (1:5000; Novus Biologicals) and mouse monoclonal anti-IRE1α antibody (1:1000; Santa Cruz Biotechnology, Inc.). The secondary antibodies used included HRP-conjugated goat anti-rabbit IgG antibody (1:2000; Santa Cruz Biotechnology, Inc.) and HRP-conjugated goat anti-mouse IgG antibody (1:2000; Santa Cruz Biotechnology), β-actin was immunoblotted with peroxidase-conjugated mouse monoclonal anti-β-actin antibody (1:50,000; Sigma). The immunostained protein bands were detected with ECL™ Chemiluminescent detection reagents (Pierce Biotechnology, Inc., Rockford, IL), and imaged by using a CL-XPosure film (Pierce Biotechnology).