Western Blot Analysis of ER Stress Markers

Protein lysates were subjected to SDS-PAGE, electrotransferred to a polyvinylidene difluoride membranes (PVDF; Merck Millipore; Billerica, MA, USA) and subsequently incubated with the following antibodies: ATF6α (90 kDa) (1:1000, sc-166659), CHOP (GADD153) (26 kDa) (1:1000, sc-7351), peIF2α (Ser52) (36 kDa) (1:1000, sc-12412), iNOS (1300 kDa) (1:1000, sc-7271) (Santa Cruz Biotechnology; Santa Cruz, CA, USA) and β-actin (42 kDa) (1:5000; A5316) (Sigma; St. Louis, MO, USA) after incubating the membranes with 3% BSA (β-actin, ATF6α), 5% BSA (peIF2α) or 5% skim milk (CHOP, iNOS) blocking buffer. Specific antigen–antibody bindings were detected using horseradish-peroxidase conjugated secondary antibodies (Dako Denmark; Glostrup, Denmark) and an enhanced chemiluminescence detection method, according to the manufacturer’s instructions (Pierce ECL Western Blotting Substrate; Thermo Scientific, Waltham, MA, USA) as described previously [127 (link),128 (link)]. Autoradiographic films (Fujifilm; Tokyo, Japan) were scanned and the band’s signal was quantified by densitometry using ImageJ-1.53 software (National Institutes of Health, Bethesda, MD, USA). Values were expressed relative to β-actin.