Journal: The Journal of Experimental Medicine

Article Title: Enhancement and suppression of signaling by the conserved tail of IgG memory–type B cell antigen receptors

doi: 10.1084/jem.20061923

Figure Lengend Snippet: IgG membrane tail does not alter tyrosine phosphorylation of CD22 or ERK phosphorylation. Splenocytes from IgM and IgMG transgenic mice were stimulated with 50 μg/ml anti-IgM F(ab′) 2 . Total cellular proteins (A) or immunoprecipitated CD22 (B) were fractionated by SDS-PAGE and Western blotted with antiphosphotyrosine antibody (A and B, top) or anti-CD22 antibody (B, bottom). The ratios of phosphorylated CD22 to total CD22 are indicated. (C and D) Mean fluorescence intensity (MFI) of permeabilized B220 + CD21 medium CD23 + follicular B cells stained by flow cytometry for phosphorylated ERK either (C) at the indicated times after stimulation with 50 μg/ml anti-IgM F(ab′) 2 or (D) in unstimulated (−) versus stimulated (+) cells after 2 min in the presence of MEK inhibitors PD98059, U0126, or DMSO as diluent controls. Data are representative of two experiments.

Article Snippet: After stripping, the membranes were stained with goat anti–mouse Siglec-2 (CD22) antibody (R&D Systems), followed by horseradish peroxidase–conjugated anti–goat antibody (Jackson ImmunoResearch Laboratories).

Techniques: Membrane, Phospho-proteomics, Transgenic Assay, Immunoprecipitation, SDS Page, Western Blot, Fluorescence, Staining, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Enhancement and suppression of signaling by the conserved tail of IgG memory–type B cell antigen receptors

doi: 10.1084/jem.20061923

Figure Lengend Snippet: IgG membrane tail evokes an augmented Ca 2+ response independent of CD22. RBC-depleted splenocytes from mice of the indicated genotypes were labeled with 1 μM Indo-1 for 30 min at 37°C. (A and B) The cells were counterstained with antibodies against B220 and CD21 for the final 10 min of Indo-1 loading. The stained cells were at first acquired for 30 s and then stimulated with the indicated concentrations of anti-IgM F(ab′) 2 antibody (A, 5 μg/ml; and B, 0.5 μg/ml). Lines show the mean Indo-1 ratio in B220 + CD21 medium cells. Data from one out of three independent experiments are shown. (C and D) Indo-1–loaded cells stained with antibody against B220 were simulated with the indicated concentrations of HEL conjugated to PE (HEL-PE; C, 5 μg/ml; and D, 0.5 μg/ml), and the Indo-1 ratio was measured on gated HEL + B220 + cells.

Article Snippet: After stripping, the membranes were stained with goat anti–mouse Siglec-2 (CD22) antibody (R&D Systems), followed by horseradish peroxidase–conjugated anti–goat antibody (Jackson ImmunoResearch Laboratories).

Techniques: Membrane, Labeling, Staining

Journal: The Journal of Experimental Medicine

Article Title: Enhancement and suppression of signaling by the conserved tail of IgG memory–type B cell antigen receptors

doi: 10.1084/jem.20061923

Figure Lengend Snippet: IgG membrane tail increases antibody production independent of CD22. 5 × 10 5 HEL-binding splenic B cells from IgMG or IgM transgenic donors of the indicated CD22 genotypes were adoptively transferred into nonirradiated C57BL/6 mice, and the recipient mice were immunized with HEL in CFA. The concentration of anti-HEL IgM a antibody 10 d after immunization was measured in the serum of individual recipient mice (circles) by ELISA. Data are representative of three separate experiments. Significant differences were determined by the Mann-Whitney test. *, P < 0.05; **, P < 0.01.

Article Snippet: After stripping, the membranes were stained with goat anti–mouse Siglec-2 (CD22) antibody (R&D Systems), followed by horseradish peroxidase–conjugated anti–goat antibody (Jackson ImmunoResearch Laboratories).

Techniques: Membrane, Binding Assay, Transgenic Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: Enhancement and suppression of signaling by the conserved tail of IgG memory–type B cell antigen receptors

doi: 10.1084/jem.20061923

Figure Lengend Snippet: CD22 deficiency suppresses IgM but not IgMG marginal zone B cell differentiation. Splenocytes were stained with HEL and combinations of antibodies against CD21/CD23/HyHEL9/B220 (A and B) or CD21/CD1d/HyHEL9/B220 (C and D). (A and C) The displayed profiles are gated on B220 + HEL-binding cells, and the percentage of B220 + HEL-binding cells in each window is shown. (B and D) Percentages of HEL-binding B cells in the marginal zone subset in individual mice (circles). Significant differences, as determined by the Student's t test, are indicated.

Article Snippet: After stripping, the membranes were stained with goat anti–mouse Siglec-2 (CD22) antibody (R&D Systems), followed by horseradish peroxidase–conjugated anti–goat antibody (Jackson ImmunoResearch Laboratories).

Techniques: Cell Differentiation, Staining, Binding Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Protein Tyrosine Phosphatase SHP-1 (PTPN6) but Not CD45 (PTPRC) Is Essential for the Ligand-Mediated Regulation of CD22 in BCR-Ligated B Cells.

doi: 10.4049/jimmunol.2100109

Figure Lengend Snippet: FIGURE 1. Ligand binding of CD22 regulates BCR ligation-induced Ca21

Article Snippet: The upper membranes were reprobed with goat anti-mouse CD22 Ab (sc-7032; Santa Cruz Biotechnology) followed by incubation with HRP-conjugated anti-goat IgG (sc-2056; Santa Cruz Biotechnology).

Techniques: Ligand Binding Assay, Ligation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Protein Tyrosine Phosphatase SHP-1 (PTPN6) but Not CD45 (PTPRC) Is Essential for the Ligand-Mediated Regulation of CD22 in BCR-Ligated B Cells.

doi: 10.4049/jimmunol.2100109

Figure Lengend Snippet: FIGURE 2. CD45 is dispensable for ligand-mediated downregulation of CD22 phosphorylation. Spleen B cells from CD451/1 (A and B) and CD45/ (A and C) mice were stimulated with anti-IgM Ab in the presence (B and C) or absence (AC) of GSC718 for indicated time. Anti-CD22 im- munoprecipitates were analyzed by Western blotting using indicated Abs. Intensities of protein bands were quantified, and ratios of phosphorylated CD22 and SHP-1 to total CD22 were calculated. One-way ANOVA fol- lowed by HolmSidak post hoc test to compare the mean values is per- formed using GraphPad Prism v. 6. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The upper membranes were reprobed with goat anti-mouse CD22 Ab (sc-7032; Santa Cruz Biotechnology) followed by incubation with HRP-conjugated anti-goat IgG (sc-2056; Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Protein Tyrosine Phosphatase SHP-1 (PTPN6) but Not CD45 (PTPRC) Is Essential for the Ligand-Mediated Regulation of CD22 in BCR-Ligated B Cells.

doi: 10.4049/jimmunol.2100109

Figure Lengend Snippet: FIGURE 4. CD22 is a substrate of SHP-1. (A) Schematic representation of the substrate-trapping mutant of SHP-1. (B) Association of phosphorylat- ed SHP-1 with the substrate-trapping mutant of SHP-1. The B cell line CH1 expressing SHP-1C-WT or a substrate-trapping mutant of SHP-1C-C/ S was stimulated with pervanadate and immunoprecipitated with anti-CD22 Ab or isotype-matched control Ab. Immunoprecipitates were analyzed by Western blotting using indicated Abs. Intensities of protein bands were quantified by ImageJ and are indicated.

Article Snippet: The upper membranes were reprobed with goat anti-mouse CD22 Ab (sc-7032; Santa Cruz Biotechnology) followed by incubation with HRP-conjugated anti-goat IgG (sc-2056; Santa Cruz Biotechnology).

Techniques: Mutagenesis, Expressing, Immunoprecipitation, Control, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Protein Tyrosine Phosphatase SHP-1 (PTPN6) but Not CD45 (PTPRC) Is Essential for the Ligand-Mediated Regulation of CD22 in BCR-Ligated B Cells.

doi: 10.4049/jimmunol.2100109

Figure Lengend Snippet: FIGURE 3. GSC839 specifically inhibits binding of CD22 to cellular li- gands. A20 cells were incubated with CD22-Fc protein, and U937 cells were in- cubated with the other indicated Siglec-Fc proteins in the presence of indicated concentrations of GSC839; inhibition of the binding of Siglec-Fc proteins to the cells by GSC839 was analyzed by flow cytometry. Percentage of mean fluores- cence intensity in the presence of GSC839 to that in the absence of sialosides is calculated as percentage binding. Percentage binding inhibition is calculated as follows: 100 (percentage binding). n = 3. Error bars show mean ± SD.

Article Snippet: The upper membranes were reprobed with goat anti-mouse CD22 Ab (sc-7032; Santa Cruz Biotechnology) followed by incubation with HRP-conjugated anti-goat IgG (sc-2056; Santa Cruz Biotechnology).

Techniques: Binding Assay, Incubation, Inhibition, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Protein Tyrosine Phosphatase SHP-1 (PTPN6) but Not CD45 (PTPRC) Is Essential for the Ligand-Mediated Regulation of CD22 in BCR-Ligated B Cells.

doi: 10.4049/jimmunol.2100109

Figure Lengend Snippet: FIGURE 5. SHP-1 is required for li- gand-mediated regulation of CD22. LPS-stimulated spleen B cells were transduced with virus vector containing SHP-1C/S or empty vector. Cells were stimulated with anti-IgM for indicated time in the presence or absence of GSC839, lysed, and immunoprecipi- tated with anti-CD22. Immunoprecipi- tates were analyzed by Western blotting using indicated Abs (A). Representative data of three experiments. Intensities of protein bands were quantified, and ra- tios of phosphorylated CD22 (B) and SHP-1 (C) to total CD22 were calculat- ed. One-way ANOVA followed by HolmSidak post hoc test to compare the mean values is performed using GraphPad Prism v. 6. **p < 0.01.

Article Snippet: The upper membranes were reprobed with goat anti-mouse CD22 Ab (sc-7032; Santa Cruz Biotechnology) followed by incubation with HRP-conjugated anti-goat IgG (sc-2056; Santa Cruz Biotechnology).

Techniques: Transduction, Virus, Plasmid Preparation, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Protein Tyrosine Phosphatase SHP-1 (PTPN6) but Not CD45 (PTPRC) Is Essential for the Ligand-Mediated Regulation of CD22 in BCR-Ligated B Cells.

doi: 10.4049/jimmunol.2100109

Figure Lengend Snippet: FIGURE 6. Model of the regulation of CD22 by endogenous ligands. CD22 clusters are formed by a2,6 sialic acid- dependent CD22CD22 homotypic interaction. (A and B) Regulation of CD22 by endogenous ligands in BCR- ligated B cells. When BCR is ligated, a sizable fraction of CD22 is phosphory- lated and recruits SHP-1. SHP-1 then dephosphorylates nearby CD22 in the clusters, resulting in negative regulation of CD22 function (A). When ligand in- teraction of CD22 is disrupted, SHP-1 recruited to CD22 dephosphorylates CD22 only inefficiently because of a lack of CD22 in the close vicinity in the absence of sialic aciddependent CD22 clusters (B). (C and D) Regulation of CD22 by endogenous ligands in unsti- mulated B cells. In the absence of BCR ligation, only a small number of CD22 is phosphorylated and recruits SHP-1. SHP-1 recruited to CD22 does not de- phosphorylate CD22 because there is no phosphorylated CD22 in the close vicinity. Therefore, a2,6 sialic acidde- pendent CD22 clustering does not downregulate CD22 function, regardless of presence (C) or absence (D) of ligand recognition of CD22. Another a2,6 sial- ic aciddependent interaction, such as interaction between CD22 and BCR, may augment CD22 function (C).

Article Snippet: The upper membranes were reprobed with goat anti-mouse CD22 Ab (sc-7032; Santa Cruz Biotechnology) followed by incubation with HRP-conjugated anti-goat IgG (sc-2056; Santa Cruz Biotechnology).

Techniques: Ligation