Las 3000 plus imager

Equal amounts of extracted protein from each mouse were prepared and assayed by SDS-PAGE as described earlier. The separated proteins were then transferred to Immobilon-P membranes (EMD-Millipore). The membrane blots were first probed with a primary antibody overnight at 4°C. The primary antibodies used included: anti-BiP/GRP78 (BD Transduction Laboratories (BDTL), 610979; 1:250); anti-mouse iNOS (BDTL, 610329; 1:4000); anti-calreticulin (BDTL, 612137; 1:5000); anti-caspase 12 (BD Pharmingen, 551430; 1:2000); anti-CHOP (Cell Signaling Technology, 5554; 1:1000); anti-GRP94 (Cell Signaling Technology, 2104; 1:1000), anti p-JNK (Santa Cruz Biotechnology, SC-6254; 1:200); anti -ATF6 (Imgenex-Biotech, IMG-273; 1:150); and anti-Ero1α (Santa Cruz Biotechnology, SC-100805; 1:200). Primary antibodies were followed with horseradish peroxidase (HRP)-conjugated rabbit anti-mouse or goat anti-rabbit antibody. Anti-GAPDH, β-actin and β-tubulin primary antibody (Sigma-Aldrich, St. Louis, MO) was used for loading control. The blots were developed by incubation with Super Signal/chemiluminescent substrate (Pierce Biotechnology, Rockford, IL) for 5 min and were assayed with a LAS-3000plus imager (Fuji). The results are reported as “normalized fold expression”, which was calculated as the chemiluminescence value of the indicated analyte divided by that of the loading control.