Goat anti erp46

For western blot analyses, subconfluent cells were washed twice with PBS and placed in fresh supplemented medium containing 60 nM thapsigargin or 2.5 µg/ml tunicamycin. Protein lysates (10–30 µg) were prepared following 6 and 24 h treatment, resolved by 10% SDS-PAGE and transferred onto nitrocellulose membranes. Primary antibodies used were goat anti-ERp46 (dilution, 1:1,000; sc-49660; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), mouse anti-GRP78 (dilution, 1:1,000; #610979, BD Pharmingen, San Diego, CA, USA) and rabbit anti-PDI antibody (dilution, 1:1,000; #2446, Cell Signaling Technology, Inc., Danvers, MA, USA). Equal protein loading was verified using mouse β-actin-specific antibody (A00702; dilution, 1;1,000; GenScript, Piscataway, NJ, USA). Secondary horseradish peroxidase-conjugated antibodies (anti-goat, sc-2378; anti-rabbit, sc-2030; anti-mouse, sc-2031; dilution, 1:1,000; Santa Cruz Biotechnology Inc.) were used. Incubation in primary and secondary antibody was carried out for 2 h at room temperature in Tris-buffered saline/0.1% Tween 20. Protein bands were visualized by enhanced chemiluminescence using Pierce ECL substrate (Thermo Fisher Scientific, Inc.) and Amersham Hyperfilm ECL film (GE Healthcare Life Sciences, Chalfont, UK).