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R&D Systems goat anti cd22
Goat Anti Cd22, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/goat+anti+cd22/Mouse+Siglec-2%2FCD22+Antibody/pmc10835075__pnas__2316446121__sapp-64-6-8
Average 94 stars, based on 2 article reviews

goat anti cd22 - by Bioz Stars, 2026-11

94/100 stars

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Incubation:

Article Title: Nutrient-derived signals regulate eosinophil adaptation to the small intestine
Article Snippet: .. This was followed by incubation with goat anti-CD22 (R&D Systems, #AF2296, 0.67 μg/mL) and rat anti-MBP (0.5 μg/mL) antibodies in the same buffer as above (without glycine) overnight at 4° C. Secondary antibody incubation, DAPI staining, and sample mounting was performed as described earlier. ..

Staining:

Image Search Results

Neu5G c is dispensable for upregulation of BCR ligation-induced signaling by ligand-CD22 interaction . Spleen B cells were obtained from wild type (WT), S t 6 g al1 −/− and Cmah −/− C57BL/6 mice. Cells were loaded with Fluo-4, and stimulated with anti-IgM antibody without ( A–D ) or with ( C and D ) GSC718. Intracellular Ca 2+ level was measured by flow cytometry. Representative data are shown ( A and C ). The arrows indicate the time point when the stimulants were added. The basal MFI was subtracted from the peak MFI ((peak - basal) MFI) ( B and D ). Normalized net MFI (GSC718(+)/GSC718(−)) was calculated by the following formula. Normalized net MFI ( GSC 718 ( + ) / GSC 718 ( − ) , % ) = ( peak − basal ) MFI with GSC 718 ( peak − basal ) MFI without GSC 718 × 100 . Data were analyzed by paired Student’s t test (n = 5 for S t 6 g al1 − /− and n = 4 for Cmah −/− ) ( B ) or one-way ANOVA followed by Tukey’s multiple comparison test (n = 8 for WT, n = 5 for S t 6 g al1 − /− , n = 4 for Cmah −/− ) ( D ). ns p > 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.001.

Journal: The Journal of Biological Chemistry

Article Title: Neu5Gc-mediated high-affinity interaction is dispensable for CD22 cis-ligands to regulate B cell signaling

doi: 10.1016/j.jbc.2024.107630

Figure Lengend Snippet: Neu5G c is dispensable for upregulation of BCR ligation-induced signaling by ligand-CD22 interaction . Spleen B cells were obtained from wild type (WT), S t 6 g al1 −/− and Cmah −/− C57BL/6 mice. Cells were loaded with Fluo-4, and stimulated with anti-IgM antibody without ( A–D ) or with ( C and D ) GSC718. Intracellular Ca 2+ level was measured by flow cytometry. Representative data are shown ( A and C ). The arrows indicate the time point when the stimulants were added. The basal MFI was subtracted from the peak MFI ((peak - basal) MFI) ( B and D ). Normalized net MFI (GSC718(+)/GSC718(−)) was calculated by the following formula. Normalized net MFI ( GSC 718 ( + ) / GSC 718 ( − ) , % ) = ( peak − basal ) MFI with GSC 718 ( peak − basal ) MFI without GSC 718 × 100 . Data were analyzed by paired Student’s t test (n = 5 for S t 6 g al1 − /− and n = 4 for Cmah −/− ) ( B ) or one-way ANOVA followed by Tukey’s multiple comparison test (n = 8 for WT, n = 5 for S t 6 g al1 − /− , n = 4 for Cmah −/− ) ( D ). ns p > 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.001.

Article Snippet: Mouse spleen cells were treated with anti-FcγRII/III antibody 2.4G2 for 15 min to block FcγRII/III-mediated nonspecific binding, and stained with the following antibodies: Anti-mouse CD22 (F239) ( ) and anti-mouse IgM (115-006-020, Jackson ImmunoResearch) conjugated with Alexa647 and Pacific Blue, respectively, using Alexa Fluor 647 and Pacific Blue antibody conjugation kits (Invitrogen), FITC-conjugated anti-mouse CD21 (7E9, BioLegend; 7G6, BD PharMingen), PE-conjugated anti-mouse CD23 (B3B4, eBioscience), biotin-conjugated anti-mouse CD23 (B3B4, BD PharMingen), R-PE-conjugated streptavidin (Invitrogen), biotin-conjugated anti-mouse CD19 (eBio13, eBioscience), Pacific Blue-conjugated streptavidin (Invitrogen), APC-conjugated anti-mouse CD45R/B220 (RA3-6B2, BD Pharmingen), FITC-conjugated anti-mouse CD45R/B220 (RA3-6B2, BD Pharmingen), Alexa647-conjugated anti-mouse IgD (11-26c, eBioscience), FITC-conjugated anti-mouse IgM (1020-02, SBA).

Techniques: Ligation, Flow Cytometry, Comparison

Surface expression of IgM and CD22 is reduced in S t 6 g al1 −/− but not Cmah −/− B cells. Spleen cells were obtained from wild-type (WT), S t 6 g al1 −/− and Cmah −/− C57BL/6 mice. A–D , B220 + IgM lo IgD hi FO B cells from WT ( A–D ), S t 6 g al1 −/− ( A and B ) and Cmah −/− mice ( C and D ) were gated and analyzed for IgM and IgD expression. Representative data are shown ( A and C ). MFIs are indicated. MFIs of IgM and IgD (n = 3) and Mean ± SD are shown ( B and D ). E-H , B220 + cells from WT ( E–H ), S t 6 g al1 − /− ( E and F ) and Cmah −/− ( G and H ) mice were gated and analyzed for CD22. Representative data are shown ( E and G ). MFIs of CD22 are indicated. MFIs of CD22 (n = 3) and Mean ± SD are shown ( F and H ). Data were analyzed by Student’s t test. ns: not significant, ∗∗∗ p < 0.005.

Journal: The Journal of Biological Chemistry

Article Title: Neu5Gc-mediated high-affinity interaction is dispensable for CD22 cis-ligands to regulate B cell signaling

doi: 10.1016/j.jbc.2024.107630

Figure Lengend Snippet: Surface expression of IgM and CD22 is reduced in S t 6 g al1 −/− but not Cmah −/− B cells. Spleen cells were obtained from wild-type (WT), S t 6 g al1 −/− and Cmah −/− C57BL/6 mice. A–D , B220 + IgM lo IgD hi FO B cells from WT ( A–D ), S t 6 g al1 −/− ( A and B ) and Cmah −/− mice ( C and D ) were gated and analyzed for IgM and IgD expression. Representative data are shown ( A and C ). MFIs are indicated. MFIs of IgM and IgD (n = 3) and Mean ± SD are shown ( B and D ). E-H , B220 + cells from WT ( E–H ), S t 6 g al1 − /− ( E and F ) and Cmah −/− ( G and H ) mice were gated and analyzed for CD22. Representative data are shown ( E and G ). MFIs of CD22 are indicated. MFIs of CD22 (n = 3) and Mean ± SD are shown ( F and H ). Data were analyzed by Student’s t test. ns: not significant, ∗∗∗ p < 0.005.

Techniques: Expressing

α2,6 sialic acid but not Neu5Gc is required for CD22 ligand-mediated restoration of BCR ligation-induced signaling in C d 45 − /− B cells. BCR ligation-induced Ca 2+ signaling in spleen B cells from wild-type (WT) C57BL/6 mice ( A – C ) or C57BL/6 mice deficient in either ST6Gal1 ( A and C ), Cmah ( B and C ), CD45 ( A–C ), both CD45 and ST6Gal1 ( A and C ) or both CD45 and Cmah ( B and C ). Cells were loaded with Fluo-4, and stimulated with anti-IgM antibody. Intracellular Ca 2+ level was measured by flow cytometry. Representative data are shown ( A and B ) The arrows indicate the time point when anti-IgM was added. The basal MFI was subtracted from the peak MFI ((peak - basal) MFI) ( C ). Normalized net MFI ( C d 45 −/− / C d 45 +/+ ) was calculated by the following formula. Normalized net MFI Cd 45 − / − / Cd 45 + / + , % = peak − basal MFI in Cd 45 − / − B cells peak − basal MFI in Cd 45 + / + B cells × 100 . Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test (n = 4 for WT and S t 6 g al1 −/− , n = 3 for Cmah −/− ). ns p > 0.05, ∗∗ p < 0.01.

Journal: The Journal of Biological Chemistry

Article Title: Neu5Gc-mediated high-affinity interaction is dispensable for CD22 cis-ligands to regulate B cell signaling

doi: 10.1016/j.jbc.2024.107630

Figure Lengend Snippet: α2,6 sialic acid but not Neu5Gc is required for CD22 ligand-mediated restoration of BCR ligation-induced signaling in C d 45 − /− B cells. BCR ligation-induced Ca 2+ signaling in spleen B cells from wild-type (WT) C57BL/6 mice ( A – C ) or C57BL/6 mice deficient in either ST6Gal1 ( A and C ), Cmah ( B and C ), CD45 ( A–C ), both CD45 and ST6Gal1 ( A and C ) or both CD45 and Cmah ( B and C ). Cells were loaded with Fluo-4, and stimulated with anti-IgM antibody. Intracellular Ca 2+ level was measured by flow cytometry. Representative data are shown ( A and B ) The arrows indicate the time point when anti-IgM was added. The basal MFI was subtracted from the peak MFI ((peak - basal) MFI) ( C ). Normalized net MFI ( C d 45 −/− / C d 45 +/+ ) was calculated by the following formula. Normalized net MFI Cd 45 − / − / Cd 45 + / + , % = peak − basal MFI in Cd 45 − / − B cells peak − basal MFI in Cd 45 + / + B cells × 100 . Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test (n = 4 for WT and S t 6 g al1 −/− , n = 3 for Cmah −/− ). ns p > 0.05, ∗∗ p < 0.01.

Techniques: Ligation, Flow Cytometry, Comparison

Neu5Gc is dispensable for down-regulation of basal Ca 2+ signaling in B cells by ligand-CD22 interaction. Spleen B cells were obtained from wild type (WT), S t 6 g al1 −/− and Cmah −/− C57BL/6 mice. Cells were loaded with Fluo-4, and stimulated with or without GSC718. Intracellular Ca 2+ level was measured by flow cytometry. Representative data are shown ( A and C ). The arrows indicate the time point when GSC718 was added. Normalized increase in MFI by GSC718 was calculated by the following formula ( B ) to correct the difference in the basal MFI between experiments. Normalized increase in MFI by GSC 718 ( % ) = highest MFI with GSC 718 − highest MFI without GSC 718 highest MFI without GSC 718 × 100 . Data were analyzed by one-way ANOVA followed by Dunnet’s multiple comparison test (n = 4). ns p > 0.05, ∗∗ p < 0.01.

Journal: The Journal of Biological Chemistry

Article Title: Neu5Gc-mediated high-affinity interaction is dispensable for CD22 cis-ligands to regulate B cell signaling

doi: 10.1016/j.jbc.2024.107630

Figure Lengend Snippet: Neu5Gc is dispensable for down-regulation of basal Ca 2+ signaling in B cells by ligand-CD22 interaction. Spleen B cells were obtained from wild type (WT), S t 6 g al1 −/− and Cmah −/− C57BL/6 mice. Cells were loaded with Fluo-4, and stimulated with or without GSC718. Intracellular Ca 2+ level was measured by flow cytometry. Representative data are shown ( A and C ). The arrows indicate the time point when GSC718 was added. Normalized increase in MFI by GSC718 was calculated by the following formula ( B ) to correct the difference in the basal MFI between experiments. Normalized increase in MFI by GSC 718 ( % ) = highest MFI with GSC 718 − highest MFI without GSC 718 highest MFI without GSC 718 × 100 . Data were analyzed by one-way ANOVA followed by Dunnet’s multiple comparison test (n = 4). ns p > 0.05, ∗∗ p < 0.01.

Techniques: Flow Cytometry, Comparison

FIGURE 1. Ligand binding of CD22 regulates BCR ligation-induced Ca21

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

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.

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.

Techniques: Phospho-proteomics, Western Blot

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.

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.

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

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.

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.

Techniques: Binding Assay, Incubation, Inhibition, Flow Cytometry

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.

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.

Techniques: Transduction, Virus, Plasmid Preparation, Western Blot

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

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).

Techniques: Ligation

Serum anti-NP responses to unsialylated (−; NP–PA) or natively sialylated (+; NP–PA–NeuGc) compounds. (A–C) Responses of (A) wild-type, (B) CD22 −/− , and (C) Siglecg −/− mice. Anti-NP titers were assessed on days 7 and 14 as indicated. (top) IgM anti-NP titers; (bottom) IgG3 anti-NP titers. Each point represents the response of an individual mouse of the indicated genotype. Results shown are representative of at least two independent experiments. Horizontal bars represent means. *, P < 0.05; and ***, P < 0.005 using the two-tailed Student’s t test. n.s., not significant.

Journal: The Journal of Experimental Medicine

Article Title: Decoration of T-independent antigen with ligands for CD22 and Siglec-G can suppress immunity and induce B cell tolerance in vivo

doi: 10.1084/jem.20091873

Figure Lengend Snippet: Serum anti-NP responses to unsialylated (−; NP–PA) or natively sialylated (+; NP–PA–NeuGc) compounds. (A–C) Responses of (A) wild-type, (B) CD22 −/− , and (C) Siglecg −/− mice. Anti-NP titers were assessed on days 7 and 14 as indicated. (top) IgM anti-NP titers; (bottom) IgG3 anti-NP titers. Each point represents the response of an individual mouse of the indicated genotype. Results shown are representative of at least two independent experiments. Horizontal bars represent means. *, P < 0.05; and ***, P < 0.005 using the two-tailed Student’s t test. n.s., not significant.

Article Snippet: Goat anti–mouse CD22 was used at 1:100 dilution in 5% milk in TBS/T (M-20; Santa Cruz Biotechnology, Inc.).

Techniques: Two Tailed Test

Analysis of tolerance induction by the sialylated antigens NP–PA–NeuGc and NP–PA–bNeuGc, and responses in siglec-deficient mice. (A–D) Functional assay of in vivo tolerance induction. Results shown are representative of at least two independent experiments. (A) Mice ( n = 4/group) were challenged with NP 65 –PA–NeuGc on day 0 and rechallenged with NP 200 –PA on day 17. IgM and IgG3 antibody levels were assessed at the indicated time points. Control mice received either NP 65 –PA on day 0 and were rechallenged with NP 200 –PA as in the experimental group, or received just NP 200 –PA without pretreatment (green lines). #, a significant reduction in NP 65 –PA–NeuGc–pretreated mice at 7 d after challenge with NP 200 –PA (P = 8.84 × 10 −5 ) compared to nonpretreated animals at 7 d after NP 200 –PA challenge (green line). (B) Mice ( n = 4/group) were challenged with NP–PA–bNeuGc on day 0 and rechallenged with NP–PA on days 14 and 31. Control mice received NP-Ficoll on day 0 and were rechallenged with NP–PA as in the experimental group. (C and D) Tolerance induction in siglec-deficient mice. Serum IgM and IgG3 anti-NP responses of mice that were primed on day 0 with either NP–PA–bNeuGc or NP–PA and boosted on days 17 or 18 with NP–PA. (C) Responses in CD22 −/− mice ( n = 8 NP–PA; n = 9 NP–PA–bNeuGc). Secondary challenge was done on day 17. Because of the known weak TI-2 responses in CD22 −/− mice, all conjugate injections were performed with a double dose of antigen (40 µg/mouse). (D) Responses in Siglecg −/− mice ( n = 8 NP–PA; n = 9 NP–PA–bNeuGc). Secondary challenge was done on day 18. Shown are means + SD. *, P < 0.05; **, P < 0.01; and ***, P < 0.005 using the two-tailed Student’s t test.

Journal: The Journal of Experimental Medicine

Article Title: Decoration of T-independent antigen with ligands for CD22 and Siglec-G can suppress immunity and induce B cell tolerance in vivo

doi: 10.1084/jem.20091873

Figure Lengend Snippet: Analysis of tolerance induction by the sialylated antigens NP–PA–NeuGc and NP–PA–bNeuGc, and responses in siglec-deficient mice. (A–D) Functional assay of in vivo tolerance induction. Results shown are representative of at least two independent experiments. (A) Mice ( n = 4/group) were challenged with NP 65 –PA–NeuGc on day 0 and rechallenged with NP 200 –PA on day 17. IgM and IgG3 antibody levels were assessed at the indicated time points. Control mice received either NP 65 –PA on day 0 and were rechallenged with NP 200 –PA as in the experimental group, or received just NP 200 –PA without pretreatment (green lines). #, a significant reduction in NP 65 –PA–NeuGc–pretreated mice at 7 d after challenge with NP 200 –PA (P = 8.84 × 10 −5 ) compared to nonpretreated animals at 7 d after NP 200 –PA challenge (green line). (B) Mice ( n = 4/group) were challenged with NP–PA–bNeuGc on day 0 and rechallenged with NP–PA on days 14 and 31. Control mice received NP-Ficoll on day 0 and were rechallenged with NP–PA as in the experimental group. (C and D) Tolerance induction in siglec-deficient mice. Serum IgM and IgG3 anti-NP responses of mice that were primed on day 0 with either NP–PA–bNeuGc or NP–PA and boosted on days 17 or 18 with NP–PA. (C) Responses in CD22 −/− mice ( n = 8 NP–PA; n = 9 NP–PA–bNeuGc). Secondary challenge was done on day 17. Because of the known weak TI-2 responses in CD22 −/− mice, all conjugate injections were performed with a double dose of antigen (40 µg/mouse). (D) Responses in Siglecg −/− mice ( n = 8 NP–PA; n = 9 NP–PA–bNeuGc). Secondary challenge was done on day 18. Shown are means + SD. *, P < 0.05; **, P < 0.01; and ***, P < 0.005 using the two-tailed Student’s t test.

Article Snippet: Goat anti–mouse CD22 was used at 1:100 dilution in 5% milk in TBS/T (M-20; Santa Cruz Biotechnology, Inc.).

Techniques: Functional Assay, In Vivo, Control, Two Tailed Test

In vitro analysis of conjugate binding and response by NP-specific B cells. (A) Flow cytometry analysis of conjugate binding to isolated NP-specific B cells from QM transgenic mice. PA ligands used in this assay carried biotin tags along with the indicated carbohydrate and hapten moieties. (B) Ca 2+ mobilization response monitored by Fluo-4 dye fluorescence. (C) Analysis of proliferation induced by the indicated concentrations of antigens by 3 [H]thymidine uptake. Cells were harvested at 39 h of culture after a 15-h labeling period. Shown are means + SD. *, P < 0.05; and ***, P < 0.005 using the two-tailed Student’s t test. (D–G) Western blot analysis of protein phosphorylation induced by the indicated conjugates. The arrow indicates the expected molecular weight of CD22 on the pTyr blot. The number of independent replicates of the experiments shown in the indicated sections were as follows: A, one; B, two; C, three; D, three; and E–G, one.

Journal: The Journal of Experimental Medicine

Article Title: Decoration of T-independent antigen with ligands for CD22 and Siglec-G can suppress immunity and induce B cell tolerance in vivo

doi: 10.1084/jem.20091873

Figure Lengend Snippet: In vitro analysis of conjugate binding and response by NP-specific B cells. (A) Flow cytometry analysis of conjugate binding to isolated NP-specific B cells from QM transgenic mice. PA ligands used in this assay carried biotin tags along with the indicated carbohydrate and hapten moieties. (B) Ca 2+ mobilization response monitored by Fluo-4 dye fluorescence. (C) Analysis of proliferation induced by the indicated concentrations of antigens by 3 [H]thymidine uptake. Cells were harvested at 39 h of culture after a 15-h labeling period. Shown are means + SD. *, P < 0.05; and ***, P < 0.005 using the two-tailed Student’s t test. (D–G) Western blot analysis of protein phosphorylation induced by the indicated conjugates. The arrow indicates the expected molecular weight of CD22 on the pTyr blot. The number of independent replicates of the experiments shown in the indicated sections were as follows: A, one; B, two; C, three; D, three; and E–G, one.

Article Snippet: Goat anti–mouse CD22 was used at 1:100 dilution in 5% milk in TBS/T (M-20; Santa Cruz Biotechnology, Inc.).

Techniques: In Vitro, Binding Assay, Flow Cytometry, Isolation, Transgenic Assay, Fluorescence, Labeling, Two Tailed Test, Western Blot, Phospho-proteomics, Molecular Weight

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