Attle, Wash.) (12). This vector bears the proximal lck P2Y2 Receptor manufacturer promoter and is active mainly in thymocytes. Transgenic mice have been produced according to established protocols by the IRCM Transgenic Service. At the very least two independent founders of each and every transgenic kind had been applied in our research. Mice lacking expression of CD45 (four) or SHP-1 (motheaten) (33) were obtained from the Jackson Laboratory, Bar Harbor, Maine. Those lacking PEP had been obtained from Matt Thomas (Washington University, St. Louis, Mo.). They had been created by replacing most of the phosphatase domain of PEP with a neomycin resistance cassette (M. Thomas, private communication). These mice lacked functional PEP protein and exhibited no obvious defect in T-cell development. Cell stimulation. Normally, thymocytes (30 106) had been stimulated for the indicated periods of time at 37 with biotinylated anti-CD3 MAb 145-2C11 (ten g) or anti-TCR H57-597 (10 g) and avidin (14 g) within a volume of 200 l. Unstimulated controls were incubated at 37 with avidin alone. Just after lysis in buffer containing maltoside (1 n-dodecyl- -D-maltoside, 50 mM Tris [pH 7.6], 150 mM NaCl, two mM EDTA) supplemented with protease and phosphatase inhibitors (13), postnuclear lysates had been processed for immunoprecipitation or immunoblotting. In some experiments, lysates had been separated by Abl Inhibitor Purity & Documentation sucrose density gradient centrifugation (see under). Immunoprecipitations and immunoblots. Unless specified, immunoprecipitations and immunoblottings were performed in accordance with previously described protocols (13, 34), with all the exception that maltoside-containing buffer was utilised. Functional assays. Utilizing magnetic columns (Stem Cell Technologies, Vancouver, British Columbia, Canada), CD4 or CD8 T cells had been purified from thymus, spleen, or lymph nodes of individual mice. The purity of the cell preparations was verified by flow cytometry and was regularly higher than 90 (information not shown). Employing anti-CD3 MAb 145-2C11 (1 or three g/ml) coated on plastic, with or without having soluble anti-CD28 MAb 37.51 (1 g/ml), T cells have been activated in vitro for 40 to 48 h. In some experiments, recombinant IL-2 (20 U/ml) was added to the culture medium. Controls have been stimulated with phorbol myristate acetate (PMA) (50 ng/ml) and ionomycin (100 ng/ml). Soon after stimulation, proliferation was measured by assaying for [3H]thymidine incorporation, although cytokine production was revealed by enzyme-linked immunosorbent assay (R D Systems, Minneapolis, Minn.). All assays have been completed in triplicate, and experiments have been repeated at the very least 3 instances. Cell fractionation. Cells (150 106) have been lysed in 1 ml of Brij 58-containing buffer (1 Brij 58, 25 mM Tris [pH 7.6], 150 mM NaCl, 5 mM EDTA) supplemented with protease and phosphatase inhibitors. Lysates had been then mixed with 1 ml of 80 sucrose (produced inside the similar buffer without the need of detergent) and overlaid sequentially with 2 ml of 30 sucrose and 1 ml of 5 sucrose. Just after centrifugation at 200,000 g for 16 h at four , 0.5-ml fractions had been collected in the top from the gradient. Typically, fractions two to four contained the lipid rafts even though fractions 7 to ten contained the soluble proteins. Person fractions were analyzed by immunoblotting or immunoprecipitation, right after solubilization employing 1 maltoside. In some situations, fractions had been pooled prior to evaluation. Intracellular calcium fluxes. Ex vivo thymocytes (two 106) had been loaded with Indo-1 (10 M; Molecular Probes, Eugene, Oreg.) for 45 min at 37 and stained for ten min at space temperature with ph.