Enhanced chemiluminescence

Western blotting was performed as previously described [16 (link)]. A bicinchoninic acid assay (#23,225) was used to measure protein concentrations (Pierce, Rockford, IL, USA). Equal amounts of protein extracts 40 µg from liver were separated by a 12% SDS-PAGE and transferred onto an immobile PVDF membrane (BIO-RAD Laboratories, Inc., Hercules, CA, USA) with transfer buffer (25 mM Tris-HCl [pH 8.9], 192 mM glycine, and 20% methanol). The membranes were cut according to molecular weight range of antibodies and incubated with primary antibodies against p-eIF2α (#3398), α, eIF2α(#2103), XBP1(#27,901), CHOP(#2895) from Cell Signalling (Danvers, MA, USA) at 1:1000, and β-actin(A5441) from Sigma-Aldrich (St. Louis, MO, USA) at 1:10000 dilution overnight at 4 °C. The membranes were washed three times, incubated with secondary anti-mouse or anti-rabbit IgG antibodies at 1:1000 dilution, and visualized using enhanced chemiluminescence (SYNGENE, Frederick, MD, USA). The relative protein presence of p-eIF2α, eIF2, XBP1 and CHOP were calculated based on the ratio of intensity of each protein bands to the corresponding β-actin. Band densities were quantified using a ImageJ Launcher .