We also thank Dr. Virus was rapidly cleared without signs of secondary replication. To improve the efficiency of MV production, the hSLAM+ mice were bred with mice Cilliobrevin D having a (transgenic mice were bred into a mice were generated and bred to homozygosity and used in subsequent experiments. Mice deficient in were obtained from the laboratory of David Levy (New York University School of Medicine, New York). Viruses and Infections. The GFP-expressing viruses MVedGFP, Cilliobrevin D MVwtfGFP, and MVedGFP-SLAMblind are described in refs. 24-26. Activated lymphocytes and DC were infected at a multiplicity of infection of 5 unless otherwise stated. Intranasal (i.n.) infections were performed similarly to methods described in ref. PRKMK6 6. For i.n. and i.p. infections, respectively, 2.5 106 PFU and 1 107 PFU were used. Isolation and Activation of Lymphocytes and DC. The lymph nodes and spleens of or C57BL/6 mice were harvested through a 0.45-m mesh in RPMI medium 1640 containing 10% FBS and 0.1% 2-mercaptoethanol. Both T and B cells were negatively selected for; purified T cells were activated by using anti-CD3 and IL-2 (50 units/ml), and purified B cells were activated by using 20 g/ml LPS. Cell activation status was analyzed 48 h after harvest. DC were harvested as described in ref. 27. On day 9, the cells in suspension were harvested, counted, analyzed by FACS, and replated in activation media (RPMI medium 1640 + 10% FBS/0.1% 2-mercaptoethanol/antibiotics/5 ng/ml GM-CSF/100 ng/ml LPS). Activation status was analyzed 24 h later. Immunoprecipitation Experiments. Tissues from dissected mice were sonicated by a polytron homogenizer in RIPA buffer (50 mM Hepes/150 mM NaCl/detergents and protease inhibitors). The lysed cells were centrifuged at 10,000 for 10-15 min at 4C, and the supernatant was recovered. Protein concentrations were measured, 10 l of anti-MV H monoclonal antibody (Chemicon) was added to equivalent amounts of protein, and the mixture was incubated overnight at 4C, followed by incubation with protein G for 1-2 h at 4C. The protein G-MV H complexes were centrifuged and washed with RIPA buffer five times. The beads were resuspended in 15 l of reducing SDS/PAGE sample buffer and boiled for Cilliobrevin D 5 min. After a 5-min spin, the supernatant was subjected to PAGE. The primary antibody was a rabbit polyclonal antibody directed to the C terminus of H protein. Immunohistochemistry. Lymph nodes and spleens were snap-frozen in OCT embedding medium (EM Science) by using liquid N2, and 10-m sections were made by using a cryoslicer. The sections were air-dried, fixed in cold acetone for 10 min, air-dried again, and rinsed in PBS. Endogenous peroxidase was blocked by using 0.3% hydrogen peroxide for 4 min. After protein blocking, the slides were incubated with a rabbit anti-MV H antibody at a dilution of 1/100 for 1 h at room temperature, washed well in PBS, and incubated with an anti-rabbit biotinylated linking antibody for 30 min at room temperature. They were then washed well in PBS, incubated with Ultra Streptavidin-Horseradish Peroxidase Complex (ID Labs, London, ON, Canada) for 30 min, and washed again in PBS. The slides were then developed with freshly prepared chromagen, washed in running tap water, and counterstained lightly with Mayer’s hematoxylin. After another wash, they were dehydrated through alcohols, cleared in xylene, and mounted in Permount (Fisher Scientific). Antibodies. Monoclonal antibodies specific for CD150 (clone A12) were purchased from BD Biosciences Pharmingen. Monoclonal antibodies recognizing H were purchased from Chemicon. A rabbit polyclonal antibody was generated against the C terminus of MV H protein. Antibodies that recognize CD11c, B220, CD4, CD8, B7.2, Iab, Gr-1, NK1.1, and CD11b were purchased from BD Biosciences. Results Generation of CD150 (SLAM) Transgenic Mice. To generate transgenic.