Ero1α

Western blotting analysis was performed as described previously [46 (link)] using primary antibodies against LCN2 (R&D Systems), ERp57, ERAP1 (Abcam; Cambridge, MA, USA), ERO1α (Novus Biologicals; Littleton, CO, USA), HO-1 (Enzo Life Sciences; Farmingdale, NY, USA), CRT, NRF2, calnexin, p47phox, β-actin (Santa Cruz Biotechnology; Dallas, TX, USA), and PDI (Cell Signaling; Danvers, MA, USA). Goat anti-rabbit-IgG-HRP (Cell Signaling), donkey anti-goat IgG (Santa Cruz Biotechnology), and rabbit anti-mouse-IgG-HRP (Calbiochem, San Diego, CA, USA) were used as secondary antibodies. The blots were quantified using an Alliance Mini 4 M (UVItec, Cambridge, UK). Western blot bands corresponding to each protein were quantified, and the intensity of each target protein was normalized to the intensity of the β-actin loading control. The normalized ratio of the control was set as 1.0 to compare target protein abundance in different sample. The normalized ratios of target proteins were used to compare target protein abundance in different samples. The normalized ratio is shown at the bottom of the blots. The normalized intensity values of three different experiments are plotted as mean ± standard deviation (SD).

Protein of cardiomyocytes or heart tissues was lysed in lysis buffer (Beyotime Biotechnology, Shanghai, CN). Protein concentrations were determined by BCA protein assay kit (Beyotime Biotechnology, Shanghai, CN). The SDS-PAGE was used to separate the samples and then transferred the samples onto the PVDF membrane. The membrane was blocked with TBST containing 5% non-fat dry milk at room temperature for 2 h, then washed with TBST for 3 times, followed by incubating the membrane with the specific primary antibody against cleaved caspase-3 (Cell Signaling, Boston, MA, 1:1000, Cat. No.: 9661), CHOP (Cell Signaling, Boston, MA, 1:1000, Cat. No.:2895), IP₃R (Abcam, MA, USA, 1:1000, Cat. No.: ab108517), ERO1α (Novus, USA, 1:1000, Cat. No.: NB100-2525), EndoA2 (Santa Cruz Biotechnology, Dallas, USA, 1:200, Cat. No.: sc365704), Bcl-2 (Proteintech, Chicago, USA, 1:1000, Cat. No.: 26593-1-AP), Bax (Proteintech, Chicago, USA, 1:1000, Cat. No.: 50599-2-lg), GRP78 (Proteintech, Chicago, USA, 1:1000, Cat. No.: 11587-1-AP) at 4 °C overnight. The next day, the membranes were incubated with secondary antibody (Cell Signaling, Boston, MA) conjugated to horseradish peroxidase for 1 h. Blots were developed using a Immobilon Western Chemiluminescent HRP Substrate kit (Millipore) and molecular band intensity was determined by densitometry with Image J program (NIH, Maryland, USA).