Nf κb p65 c22b4

MEF were grown in 6-well plates to confluency and treated for up to 4 hrs with HSL-C12 or 1 hr with 1 μM thapsigargin and then lysed in M-PER mammalian protein extraction reagent (Pierce, Rockford, IL) containing 5 μg/ml leupeptin, 5 μg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride, and 50 nM calyculin A. Protein sample concentrations were determined with Bradford reagent (Bio-Rad, Hercules, CA). Immunoblot analysis was performed by first separating protein (10 to 50 μg/lane) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferring it to nitrocellulose membranes. Individual gels with identical loading were run side by side when multiple primary antibodies were utilized. Membranes were blocked (5% nonfat dried milk) in 20 mM Tris-HCl (pH 7.5)−150 mM NaCl-0.1% Tween 20 for 1 hr and then incubated with specific antibodies overnight. Primary antibodies (diluted 1:1,000 in blocking buffer) for phosphoS51- eI-F2α (119A11), eI-F2α (9722), IκBα (L35A5), and NF-κB p65 (C22B4) were acquired from Cell Signaling (Danvers, MA). Binding of primary antibodies was visualized by enhanced chemiluminescence with horseradish peroxidase-conjugated secondary antibodies (1:2,500 in blocking buffer) and Renaissance Chemiluminescence Reagent Plus (Perkin-Elmer Life Sciences). Quantitation was performed with ImageJ (National Institutes of Health, Bethesda, MD).