Journal:

Article Title: The paired Ig-like receptor PIR-B is an inhibitory receptor that recruits the protein-tyrosine phosphatase SHP-1

doi:

Figure Lengend Snippet: Inhibition of BCR-induced Ca2+ mobilization by coligation with PIR molecules. Mouse splenic B cells loaded with INDO-1 dye were analyzed by flow cytometry for intracellular Ca2+ levels in the presence (A) or absence (B) of extracellular Ca2+. Cells were stimulated with biotinylated F(ab′)2 fragments of anti-μ and anti-PIR mAbs either alone or in combination as indicated by arrows. Biotin (10 μg) was used as a cross-linker. Independent ligation with the anti-PIR mAb had no inhibitory effect on the anti-μ induced Ca2+ response (not shown).

Article Snippet: Polyclonal antibodies included F(ab′) 2 fragments of goat anti-mouse Ig antibody (GAM), F(ab′) 2 fragments of donkey anti-mouse Ig antibodies lacking cross-reactivity to rat Ig (DAM), F(ab′) 2 fragments of donkey anti-rat Ig antibodies lacking cross-reactivity to mouse Ig (DAR) (Immunotech, Luminy, France), and biotin-labeled F(ab′) 2 fragments of goat anti-mouse μ-chain antibodies (Southern Biotechnology Associates).

Techniques: Inhibition, Flow Cytometry, Ligation

Journal:

Article Title: The paired Ig-like receptor PIR-B is an inhibitory receptor that recruits the protein-tyrosine phosphatase SHP-1

doi:

Figure Lengend Snippet: Inhibition of FcɛRI-dependent cell activation by co-engagement of FcγRIIB/PIR-B chimeric molecules. (A) Assay of serotinin release by FcγRIIB/PIR-B.H cells (•) and mock transfectant cells (○). Cells were stimulated with varying dilutions of mouse IgE-producing hybridoma supernatants and the 2.4G2 anti-FcγRII/III mAb (20 μg/ml). Wild-type RBL cells yielded similar results to the mock RBL transfectant (not shown). Results expressed as mean ± SD of three independent experiments. (B) Analysis of serotonin release after co-aggregation (striped bars) versus independent aggregation (black bars) of FcγRIIB/PIR-B chimeric molecules and FcɛRI. Co-aggregation was induced by incubation of the cells with the 2.4G2 anti-FcγRII/III mAb (20 μg/ml), mouse IgE (10−3 dilution), and F(ab′)2 GAM (50 μg/ml). Independent aggregation was induced by incubation of the cells with the 2.4G2 mAb (20 μg/ml) plus F(ab′)2 DAM (100 μg/ml) and rat IgE plus F(ab′)2 DAR (100 μg/ml). Percentage inhibition was defined as the serotonin release (in cpm) induced by mouse IgE stimulation and 2.4G2 minus the background release divided by the serotonin stimulation induced by mouse IgE stimulation minus the background release. Results are representative of three independent experiments. (C) Comparative analysis of the inhibitory functions of FcγRIIB/PIR-B mutants versus wild-type chimeras. Inhibition percentages were calculated according to the above formula. FcγRIIB/PIR-B.H, FcγRIIB/PIR-B.Y794F, and FcγRIIB/PIR-B.Y824F transfected RBL cells expressed comparative levels of chimeric molecule; FACS analysis indicated mean fluorescence intensities for 2.4G2 anti-FcγR staining of 138, 165, and 89 for FcγRIIB/PIR-B.H, FcγRIIB/PIR-B.Y794F, and FcγRIIB/PIR-B.Y824F, respectively. FcγRIIB/PIR-B.L and FcγRIIB/PIR-BY794F,Y824F have similar but lower 2.4G2 fluorescence staining intensities, being 59 and 44, respectively, for FcγRIIB/PIR-B.L and FcγRIIB/PIR-B.Y794F,Y824F. The difference in inhibition exerted by FcγRIIB/PIR-B versus the double mutant was confirmed in two additional experiments (P < 0.01).

Article Snippet: Polyclonal antibodies included F(ab′) 2 fragments of goat anti-mouse Ig antibody (GAM), F(ab′) 2 fragments of donkey anti-mouse Ig antibodies lacking cross-reactivity to rat Ig (DAM), F(ab′) 2 fragments of donkey anti-rat Ig antibodies lacking cross-reactivity to mouse Ig (DAR) (Immunotech, Luminy, France), and biotin-labeled F(ab′) 2 fragments of goat anti-mouse μ-chain antibodies (Southern Biotechnology Associates).

Techniques: Inhibition, Activation Assay, Transfection, Incubation, Fluorescence, Staining, Mutagenesis

Journal:

Article Title: The paired Ig-like receptor PIR-B is an inhibitory receptor that recruits the protein-tyrosine phosphatase SHP-1

doi:

Figure Lengend Snippet: Analysis of phosphatase binding of candidate ITIMs in the PIR-B cytoplasmic domain (A) PIR-B tyrosine-containing peptides were incubated with lysates prepared from metabolically 35S-labeled IIA1.6 B cells, and adsorbed molecules were separated under reducing conditions on an 8% SDS-PAGE. (B) Binding potential of phosphorylated PIR-B peptides to the GST-SHP 1.SH2(NC), GST-SHP 2.SH2(NC), and GST-SHIP.SH2 GST fusion proteins (150 nM) was analyzed with a BIAcore apparatus. The flow rate was constant at 10 μl/min. In this experiment, 80 resonance units of phosphorylated peptides were immobilized on streptavidin-coated sensorchips. The regeneration was performed using HBS buffer supplemented with 0.02% SDS. Results expressed as corrected resonance units (CRU) corresponding to raw values after subtraction of background RU values due to the injection medium. (C) Analysis of tyrosine phosphorylation induced by co-aggregation and independent aggregation of FcɛRI and the FcγRIIB/PIR-B chimera. Cell lysates of transfected RBL cells untreated (lane 1) or treated with the 2.4G2 anti-FcγR mAb (lane 2), mouse IgE (lane 3), or both 2.4G2 and mouse IgE (lane 4) followed by F(ab′)2 GAM were subjected to immunoprecipitation with protein-G beads. The bound materials were either separated on an SDS-8% PAGE, transferred onto membranes, and immunoblotted with anti-SHP-1 (Upper, 45 × 106 FcγRIIB/PIR-B.H cells per lane) or separated on an SDS-10% PAGE, transferred onto nitrocellulose membranes, and immunobotted with anti-phosphotyrosine antibodies (Lower, 5 × 106 FcγRIIB/PIR-B.H cells per lane). The experiment shown is representative of four independent experiments.

Article Snippet: Polyclonal antibodies included F(ab′) 2 fragments of goat anti-mouse Ig antibody (GAM), F(ab′) 2 fragments of donkey anti-mouse Ig antibodies lacking cross-reactivity to rat Ig (DAM), F(ab′) 2 fragments of donkey anti-rat Ig antibodies lacking cross-reactivity to mouse Ig (DAR) (Immunotech, Luminy, France), and biotin-labeled F(ab′) 2 fragments of goat anti-mouse μ-chain antibodies (Southern Biotechnology Associates).

Techniques: Binding Assay, Incubation, Metabolic Labelling, Labeling, SDS Page, Injection, Transfection, Immunoprecipitation