Journal: Oncotarget

Article Title: Besides an ITIM/SHP-1-dependent pathway, CD22 collaborates with Grb2 and plasma membrane calcium-ATPase in an ITIM/SHP-1-independent pathway of attenuation of Ca 2+ i signal in B cells

doi: 10.18632/oncotarget.9794

Figure Lengend Snippet: A. showed CD22′s cytoplasmic tail of 113 amino acids and among them six tyrosines (Y1~Y6) and Grb2 binding motif were marked in capital letters. B. showed a list of CD22-WT and mutant forms (F, phenyalanine; N, asparagine; D, aspartate) constructs that were cloned into pApuro vector used in this study. C. showed the similar expression levels of all these CD22-WT and a series of mutant forms in DT40 cells.

Article Snippet: The Anti-mouse CD22 antibody were purchased from BD (BD Biosciences Pharmingen) [ ].

Techniques: Binding Assay, Mutagenesis, Construct, Clone Assay, Plasmid Preparation, Expressing

Journal: Oncotarget

Article Title: Besides an ITIM/SHP-1-dependent pathway, CD22 collaborates with Grb2 and plasma membrane calcium-ATPase in an ITIM/SHP-1-independent pathway of attenuation of Ca 2+ i signal in B cells

doi: 10.18632/oncotarget.9794

Figure Lengend Snippet: Parameters of the lgM-Induced Intracellular Ca 2+ i Signals in DT40 cells

Article Snippet: The Anti-mouse CD22 antibody were purchased from BD (BD Biosciences Pharmingen) [ ].

Techniques:

Journal: Oncotarget

Article Title: Besides an ITIM/SHP-1-dependent pathway, CD22 collaborates with Grb2 and plasma membrane calcium-ATPase in an ITIM/SHP-1-independent pathway of attenuation of Ca 2+ i signal in B cells

doi: 10.18632/oncotarget.9794

Figure Lengend Snippet: DT40 cells were stably transfected with empty vector ( CD22 ), CD22-WT , and a series of the mutant forms, as indicated. Cells with equal levels of membrane IgM and expressed CD22 were stimulated with 1 μg/ml F(ab’)2 anti-chicken IgM ( arrows ) for 2.5 min. In A. - D. , the Ca 2+ i responses were measured and compared among cells in the presence of 1 mM EGTA. In E , the cells were lysed for IP assay. The panel at top demonstrates IP of CD22 and IB with phorphotyrosine antibody, the second panel showed loading control blot with anti-CD22 antibody, the third panel illustrated IP of SHP-1 and IB with anti-CD22 antibody, and the panel at bottom showed loading control blot with anti-SHP-1 antibody. In F , the cells were lysed for IP assay. The upper panel showed IP of CD22 and IB with anti-PMCA antibody and the lower panel shows loading control blot with anti-CD22 antibody. Results are representative of at least three independent experiments.

Article Snippet: The Anti-mouse CD22 antibody were purchased from BD (BD Biosciences Pharmingen) [ ].

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Mutagenesis

Journal: Oncotarget

Article Title: Besides an ITIM/SHP-1-dependent pathway, CD22 collaborates with Grb2 and plasma membrane calcium-ATPase in an ITIM/SHP-1-independent pathway of attenuation of Ca 2+ i signal in B cells

doi: 10.18632/oncotarget.9794

Figure Lengend Snippet: DT40 cells were stably transfected with empty vector ( CD22 ), CD22-WT, and a series of mutant forms, as indicated. Cells with equal levels of membrane IgM and expressed CD22 were stimulated with 1 μg/ml F(ab’)2 anti-chicken IgM ( arrows ) for 2.5 min. In A. , B. , D. , and E. , the Ca 2+ i responses were measured and compared among cells in the presence of 1 mM EGTA. C. and F. compared the Ca 2+ i efflux. In G. , the cells were lysed for IP assay. The panel at top demonstrates IP of CD22 and IB with phorphotyrosine antibody, the second panel shows loading control blot with anti-CD22 antibody, the third panel IP of SHP-1 and IB with anti-CD22 antibody, and the panel at bottom shows loading control blot with anti-SHP-1 antibody. In H. , the cells were lysed for IP assay. The upper panel demonstrates IP of CD22 and IB with anti-PMCA antibody and the lower panel shows loading control blot with anti-CD22 antibody. Results are representative of at least three independent experiments.

Article Snippet: The Anti-mouse CD22 antibody were purchased from BD (BD Biosciences Pharmingen) [ ].

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Mutagenesis

Journal: Oncotarget

Article Title: Besides an ITIM/SHP-1-dependent pathway, CD22 collaborates with Grb2 and plasma membrane calcium-ATPase in an ITIM/SHP-1-independent pathway of attenuation of Ca 2+ i signal in B cells

doi: 10.18632/oncotarget.9794

Figure Lengend Snippet: Empty vector ( CD22 ) or CD22-WT ( CD22 ) was stably transfected into Grb2 −/− DT40 cells A. - D. Grb2 −/− DT40 cells were further rescued by transfection of chicken Grb2 ( B , D ), with or without co-transfection of CD22. The Ca 2+ i ( A , B ) and Ca 2+ i efflux ( C , D ) were measured in IgM-matched cells after stimulation with 2 μg/ml anti-chicken IgM ( arrows ) in the presence of 1 mM EGTA ( A , B ) and in the efflux buffer ( A , B ). Results were representative of at least three independent experiments.

Article Snippet: The Anti-mouse CD22 antibody were purchased from BD (BD Biosciences Pharmingen) [ ].

Techniques: Plasmid Preparation, Stable Transfection, Transfection, Cotransfection

Journal: Oncotarget

Article Title: Besides an ITIM/SHP-1-dependent pathway, CD22 collaborates with Grb2 and plasma membrane calcium-ATPase in an ITIM/SHP-1-independent pathway of attenuation of Ca 2+ i signal in B cells

doi: 10.18632/oncotarget.9794

Figure Lengend Snippet: In A. , CD22-WT was stably transfected into Grb2 −/− DT40 cells (the 1 st ~ 4 th lane), with (the 3 rd and 4 th lane) or without (the 1 st and 2 nd lane) co-transfection of chicken Grb2. After with (+) or without (−) stimulation of 1 μg/ml F(ab’)2 anti-chicken IgM for 2.5 min, the cells were harvested for IP study. The upper panel demonstrates IP of PMCA and IB of anti-CD22 antibody, the middle panel shows IB of Grb2, and the lower panel shows loading control blot with anti-PMCA antibody. In A. , the Grb2 −/− (the 1 st lane) or normal (the remaining lanes) DT40 cells were transfected with CD22-WT or mutant forms, as indicated. All cells were stimulated with 5 μg/ml F(ab’)2 IgM for 2.5 min and harvested for IP study. The upper panel demonstrates IP of Grb2 and IB of CD22. The middle panel shows IB of PMCA. The lower panels show the loading control blot with anti-Grb2 antibody. In C. , DT40 cells were stably transfected with Empty vector ( CD22- ), CD22-WT , with (+) or without (−) stimulation of 1 μg/ml F(ab’)2 IgM for 2.5 min. Cells lysate were obtained for IP experiment. The upper panel showed IP with PMCA antibody or normal mouse IgG and IB of Grb2. The lower panel showed loading control blot with anti-PMCA antibody.

Article Snippet: The Anti-mouse CD22 antibody were purchased from BD (BD Biosciences Pharmingen) [ ].

Techniques: Stable Transfection, Transfection, Cotransfection, Mutagenesis, Plasmid Preparation

Journal: Oncotarget

Article Title: Besides an ITIM/SHP-1-dependent pathway, CD22 collaborates with Grb2 and plasma membrane calcium-ATPase in an ITIM/SHP-1-independent pathway of attenuation of Ca 2+ i signal in B cells

doi: 10.18632/oncotarget.9794

Figure Lengend Snippet: In A. and B. , Daudi cells were transfected with scramble oligonucleotides and specific siRNA-1 and −2 towards CD22. Between 72~96 hours after transfection, cells were used for experiments. In A , cells were lysed for checking expression of CD22 ( top ), Grb2 ( middle ), and PMCA ( bottom panel ). In B. , Ca 2+ i responses were measured and compared in the presence of 1 mM EGTA after treatment of 5 μg/ml F(ab’)2 anti-human IgM ( arrows ). In C. , freshly-isolated normal human B cells (see Material and Methods ) were lysed for IP assay, with ( + ) or without ( - ) pretreatment of 5 μg/ml F(ab’)2 anti-human IgM (+). Data showed IP of CD22 ( left ), Grb2 ( middle ), and PMCA ( right ), and IB three of them as indicated. Results were representative of at least three independent experiments.

Article Snippet: The Anti-mouse CD22 antibody were purchased from BD (BD Biosciences Pharmingen) [ ].

Techniques: Transfection, Expressing, Isolation

Journal: PLoS ONE

Article Title: A Novel Epitope from CD22 Regulates Th1 and Th17 Cell Function in Systemic Lupus Erythematosus

doi: 10.1371/journal.pone.0064572

Figure Lengend Snippet: (A) The immunofluorescence assay for the combination of different Abs to mouse CD22 molecular on B cells from MRL/lpr mice. The purified B cells were stained with anti-mouse CD19 Abs (green) and anti-B2249/B2267/B2285/KLH Abs (red) separately (magnification×400). The double staining of B cells (yellow) indicated the different binding affinity of four Abs to mouse CD22. (B) Western blotting analysis indicated the specific recognition of anti-B2249/B2267/B2285/KLH Abs for the CD22 protein (135 Kd) extracted from B cells. (C) Western blotting analysis indicated that anti-B2285 Abs could recognize the whole B cell lysate merely at CD22 protein site (135Kd), and did not bind to proteins from CD4 + T cell lysate.

Article Snippet: Briefly, purified B cells were fixed with 4% paraformaldehyde at 4°C for 30 min and then incubated with the anti-B2285 Abs (1:1000) overnight, followed by FITC-conjugated anti-mouse CD19 Abs (Biolegend, California, USA) and PE-conjugated anti-mouse CD22 (clone: 2D6, eBioscience, California, USA) for 15 min at 4°C.

Techniques: Immunofluorescence, Purification, Staining, Double Staining, Binding Assay, Western Blot

Journal: PLoS ONE

Article Title: A Novel Epitope from CD22 Regulates Th1 and Th17 Cell Function in Systemic Lupus Erythematosus

doi: 10.1371/journal.pone.0064572

Figure Lengend Snippet: (A) The flow cytometry analysis for the combination of anti-B2285 Abs to mouse B cells from MRL/lpr mice in PBS, anti-KLH Abs, anti-B2285 Abs and NB2285 (B2295 peptide neutralization Abs). (B) The analysis for the combination of anti-B2285 Abs to mouse CD4 + T cells from MRL/lpr mice by flow cytometry. (C) The detection of CD22 internalization mediated by preincubated with different Abs on viable B cells by flow cytometry. (D) The assay for the competitive binding to fixed B cells between anti-B2285 Abs and commercial anti-mouse CD22 Abs. The percentage of CD19 + CD22 + B cells was decreased obviously in B2285 group compared with those in PBS and KLH groups.

Article Snippet: Briefly, purified B cells were fixed with 4% paraformaldehyde at 4°C for 30 min and then incubated with the anti-B2285 Abs (1:1000) overnight, followed by FITC-conjugated anti-mouse CD19 Abs (Biolegend, California, USA) and PE-conjugated anti-mouse CD22 (clone: 2D6, eBioscience, California, USA) for 15 min at 4°C.

Techniques: Flow Cytometry, Neutralization, Binding Assay

Journal: Oncotarget

Article Title: Ectopic expression of a novel CD22 splice-variant regulates survival and proliferation in malignant T cells from cutaneous T cell lymphoma (CTCL) patients

doi:

Figure Lengend Snippet: A . RT-PCR analysis of CD22 expression using primers amplifying exon 11-14 region of CD22, B . Western blot analysis of CD22 protein expression using FPC1 anti-CD22 mAb, C . and D . Flow cytometry analysis of CD22 cell surface expression using PE-conjugated anti-CD22 mAbs (S-HCL-1, RFB-4) or unconjugated anti-CD22 mAbs (RFB-4, FR10B4) followed by PE-conjugated secondary antibody (rabbit anti-mouse F(ab') 2 ).

Article Snippet: FR10B4, FPC1 and PE-conjugated or unconjugated RFB-4 mouse anti-CD22 monoclonal antibodies (mAbs) as well as negative control Q13 mouse anti-IL-12A p35 mAb were purchased from Santa Cruz Biotech. (Santa Cruz, CA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Flow Cytometry

Journal: Oncotarget

Article Title: Ectopic expression of a novel CD22 splice-variant regulates survival and proliferation in malignant T cells from cutaneous T cell lymphoma (CTCL) patients

doi:

Figure Lengend Snippet: A . Schematic representation of CD22 mRNA and protein structure, B . Exon-specific RT-PCR analysis of CD22 expression using primers amplifying exons 4-5 and/or exon 11-14 regions. C. RT-PCR analysis of CD22 expression using primers amplifying region before or after exon 5.

Article Snippet: FR10B4, FPC1 and PE-conjugated or unconjugated RFB-4 mouse anti-CD22 monoclonal antibodies (mAbs) as well as negative control Q13 mouse anti-IL-12A p35 mAb were purchased from Santa Cruz Biotech. (Santa Cruz, CA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Oncotarget

Article Title: Ectopic expression of a novel CD22 splice-variant regulates survival and proliferation in malignant T cells from cutaneous T cell lymphoma (CTCL) patients

doi:

Figure Lengend Snippet: MyLa2059 and MAC-2A cell lines were transfected with either non-targeting (−) or CD22-targeting (+) siRNA and cell proliferation were evaluated 24 hours after transfection. Results are representative of 5 independent experiments. Significant differences between non-targeting and CD22-targeting siRNA treated cells were evaluated using a paired two-tailed Student's t-test.

Article Snippet: FR10B4, FPC1 and PE-conjugated or unconjugated RFB-4 mouse anti-CD22 monoclonal antibodies (mAbs) as well as negative control Q13 mouse anti-IL-12A p35 mAb were purchased from Santa Cruz Biotech. (Santa Cruz, CA, USA).

Techniques: Transfection, Two Tailed Test

Journal: Oncotarget

Article Title: Ectopic expression of a novel CD22 splice-variant regulates survival and proliferation in malignant T cells from cutaneous T cell lymphoma (CTCL) patients

doi:

Figure Lengend Snippet: A . RT-PCR analysis of CD22 expression in CTCL lesional skin from patients. B . Double-staining with anti-CD4 (PE) and anti-CD22 (FITC) of biopsies from tumor stage of two MF patients.

Article Snippet: FR10B4, FPC1 and PE-conjugated or unconjugated RFB-4 mouse anti-CD22 monoclonal antibodies (mAbs) as well as negative control Q13 mouse anti-IL-12A p35 mAb were purchased from Santa Cruz Biotech. (Santa Cruz, CA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Double Staining