FRET was measured by monitoring excitation at 280 nm and emission at 450 nm, and normalized to total AMCA fluorescence (excitation at 350 nm and emission at 450 nm). Complete digestion of HLA-DR1 during the reaction was verified by SDS-PAGE analysis and silver staining of recovered reaction mixtures. Samples were boiled after the addition of 8 × Laemmli SDS-PAGE sample buffer with 5% v/v 2-mercaptoethanol, run on 12% SDS-PAGE gels, and transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Freiburg, Germany). Membranes were blocked for 1 hr in blocking buffer (1× PBS, 0·05% Tween 20 and 1× Rotiblock; Roth, Karlsruhe, Germany) and incubated for an

additional hour with the HLA-DR-specific antiserum CHAMP32 diluted in blocking buffer. After washing in PBS with 0·05% Tween 20, horseradish peroxidase (HRP)-conjugated secondary antibody (donkey Ceritinib cost anti-rabbit immunoglobulin; GE Healthcare) was diluted 1 : 5000 in blocking buffer and HM781-36B concentration incubated for 1 hr. Following additional washes, HRP activity was revealed using an enhanced chemiluminescence (ECL) detection kit (GE Healthcare) and visualized using Hyperfilm ECL (GE Healthcare). Cathepsin G−/− (CG−/−) mice on a C57BL/6 background were received from the laboratory of C. Pham (Department of Internal Medicine, Washington University School of Medicine, Saint Louis,

MO). C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were bred and maintained at the Stanford University Research Animal not Facility. The handling of all mice followed guidelines and requirements established by the National Institutes of Health and Stanford University animal research committee. Mice were killed with compressed CO2 gas and by cervical dislocation, and spleens were removed. Single-cell suspensions were prepared by mechanical disruption of the spleen through a 70-μm filter. Spleens were then treated with 1 × red blood cell (RBC) lysis buffer [1·68 m NH4Cl, 0·10 m potassium bicarbonate and 1 mm ethylenediaminetetraacetic acid (EDTA)], washed twice, and used directly for analysis. The following

antibodies were purchased from BD Biosciences (San Jose, CA): anti-mouse I-Ab phycoerythrin (PE), anti-mouse CD11b PE-Cy7, anti-mouse CD11c allophycocyanin (APC), and anti-mouse CD19 APC-Cy7. Anti-mouse CD3 Pacific Blue, anti-mouse CD45R (B220) fluorescein isothiocyanate (FITC), and anti-mouse F4/80 PerCP-Cy5.5 were purchased from eBiosciences (San Diego, CA). Before staining, cell preparations were blocked with 3·3 μg/ml anti-mouse CD16/CD32 (Fc Block; BD Biosciences) in PBS containing 0·5% bovine serum albumin (BSA) and 0·1% NaN3 for 15 min. For intracellular staining, cells were permeabilized with Cytofix/Cytoperm (BD Biosciences) for 20 min on ice, and washed with 1 × Perm/Wash Buffer (BD Biosciences).