( em C /em ) Wild-type SWAP-70 and mutants 1C525 and W297A localization before (?Ig) and after (+Ig) crosslinking of the BCR. of the cDNA was determined by 5 quick amplification of cDNA ends (CLONTECH). Immunohistochemistry. Immunostaining was performed on 4-m formalin-fixed, paraffin-embedded cells sections. Sections were deparaffinized and rehydrated, and the antigen was retrieved by using Declere (Cell Marque, Austin, TX) relating to manufacturer’s protocol. Slides were bar-coded for use within the Ventana Gen II automated immunostainer. Staining was performed according to the Ventana diaminobenzidine staining and detection BMS-214662 protocol. Slides were counterstained with hematoxylin, dehydrated in ethanol, and mounted with Permount (Fisher). Immunofluorescence. Cytospins were fixed in 3% paraformaldehyde answer for 15 min at space heat and rinsed in PBS. Cells were permeabilized by incubating for 5 min in staining buffer (PBS comprising 5% goat serum, 0.1% Triton X-100, and 0.1% sodium azide). The 1st antibody was diluted to the final concentration in staining buffer, applied onto cells, and incubated for 1 h. After three washes of 5 min each in staining buffer, the secondary antibody was applied and incubated for 30 min. Cytospins were washed three times in staining buffer, one time in PBS, and a final time in tap water, and mounted in mounting medium (Fluoromount-G; Southern Biotechnology Associates). Human being Peripheral Blood Lymphocyte Activation. Human being purified peripheral blood lymphocytes were triggered as previously explained (8). Briefly, human being peripheral blood from healthy volunteers was fractionated through Ficoll (Amersham Pharmacia), and lymphocytes in the interphase were recovered. BMS-214662 Contaminating monocytes and natural killer cells were depleted by incubating the cells with a mixture comprising anti-human CD14 and CD16 monoclonal antibodies (PharMingen), followed by immunomagnetic beads (Dynal, Great Neck, NY). Purified B and T lymphocytes were plated in activating medium [RPMI medium 1640 supplemented with 100 models/ml penicillin, 100 g/ml streptomycin, 10% FCS, 50 M 2-mercaptoethanol, 20 g/ml human being transferrin (GIBCO), 1 g/ml anti-CD2 antibody clone 6G4 and 1 g/ml anti-CD2 BMS-214662 antibody clone 4B2, 3 ng/ml IL-2 (Roche Molecular Biochemicals), BMS-214662 and 50 ng/ml IL-4 (GIBCO)] at 5,000 cells in 200 l per well inside a round-bottom 96-well plate. Cultures were tested for class switching to IgE by an IgE ELISA at day time 9 of activation. For activation of purified Igfbp2 B lymphocytes, T cells were depleted by incubating the mononuclear cells after Ficoll fractionation with a mixture of anti-CD3, anti-CD4, and anti-CD8 antibodies, followed by immunomagnetic beads. Purified B lymphocytes were plated in BMS-214662 activating medium (RPMI medium 1640 supplemented with 100 models/ml penicillin, 100 g/ml streptomycin, 10% FCS, 50 M 2-mercaptoethanol, 20 g/ml human being transferrin, 1 g/ml anti-CD40, and 50 ng/ml IL-4) at 5,000 cells in 200 l per well inside a round-bottom 96-well plate. Cocapping. For cocapping experiments, cells were incubated with the appropriate antibody at 37C for 30 min, without the use of sodium azide. Cells were then washed, centrifuged on a slide, fixed in 3% paraformaldehyde, and stained for SWAP-70. Coimmunoprecipitation. Lipopolysaccharide (LPS; for 30 min to pellet the microsomal portion. The cleared cytosolic portion was recovered, and the membrane pellet was dissolved in 400 l of hypotonic buffer comprising 0.5% SDS. Mutagenesis. Green fluorescent protein (GFP) cDNA (CLONTECH) was fused in-frame upstream of wild-type SWAP-70 by standard cloning methods. PCR primers encoding N- or C-terminally truncated proteins were designed and used to create SWAP-70 mutants fused to GFP (including 1C525, 1C312, and 1C312 NLS) and cloned into the retroviral manifestation vector pGfus. In the 1C312 NLS construct, the NLS (291C294 KKKK) was.