Western Blotting for ER Stress Markers

Western blotting procedures has been described previously [46 (link)]. The primary antibodies used were NCOA3 (Cell signalling, Cat# 2126), ATF6 (Abcam, Cat# ab122897), spliced XBP1 (Biolegend, Cat# 619502), PERK (Cell signalling, Cat# C33E10), phospho-eIF2α (Cell signalling, Cat# 9721), total eIF2α (Cell signalling, Cat# 9722), ATF4 (Santa Cruz Biotechnology, Cat# sc-200), cleaved caspase-3 (Cell Signalling, Cat# 9661) and or β-Actin (Sigma, Cat# A-5060) overnight at 4°C. The membrane was washed 3 times with PBS-0.05% Tween or TBS-0.1% Tween (total-eIF2α and phospho-eIF2α) where appropriate and further incubated in appropriate horseradish peroxidase-conjugated secondary antibody (Pierce) for 2h at room temperature. Signals were detected using Western Lightening Plus ECL (PERKin Elmer).