Anti protein disulfide isomerase

Equivalent amounts of protein determined by bicinchoninic acid protein assay kit were resolved by NuPAGE 4%-12% Bis-Tris gel (ThermoFisher Scientific) electrophoresis and blotted to Hybond-P polyvinylidene difluoride membranes (GE Healthcare Bio-Sciences, Pittsburgh, PA). Blots were blocked with 5% nonfat dry milk in Tris-saline buffer (pH 7.4) containing 0.1% Tween-20 and then probed with the following primary antibodies: anti-CL CASP3 1:250 (#9664, Cell Signaling Technology, Danvers, MA) and anti-iPLA2 1:500 (Cat 07-169-I; Millipore, Burlington, MA). Immunoreactivity was detected using horseradish peroxidase-conjugated secondary antibody (1:5000), and the bands were visualized using an enhanced chemiluminescence detection system (ThermoFisher Scientific). The membranes were probed with anti-nuclear receptor coactivator 3 (for nuclear protein; 1:5000, #PA1-845; ThermoFisher Scientific) or anti-protein disulfide isomerase (for cytoplasmic protein; 1:1000, #3501 Cell Signaling Technology) to quantify the relative protein expression level. Images of the bands were scanned and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD) for relative optical density.