Journal: Immunology

Article Title: Ligation of human Fc receptor like-2 by monoclonal antibodies down-regulates B-cell receptor-mediated signalling

doi: 10.1111/imm.12311

Figure Lengend Snippet: Reactivity of anti- Fc receptor like-2 (FCRL2) monoclonal antibodies (mAbs) with FCRL2 and other members of the FCRL family. (a) Western blot reactivity profile of anti-FCRL2 mAbs (3D8-G8) with recombinant FCRL2 protein. Lane 1: lysate of FCRL2-transfected Chinese hamster ovary (CHO) cells (15 μg); lane 2: lysate of CHO cells transfected with empty pCMV6-Neo (15 μg); lane 3: prokaryotic recombinant extracellular domain of FCRL2 (5 μg); M: protein size marker. (b) Flow cytometry analysis of the anti-FCRL2 mAbs using stable FCRL-transfected and empty vector transfected CHO cell lines. Shaded histograms represent background staining with an isotype-matched negative control mAb. 7G7 refers to a positive control commercial mAb obtained from Genentech Co. (c) ELISA results obtained for anti-FCRL2 mAbs showing specific binding to recombinant FCRL2 protein with no cross-reactivity to eukaryotic recombinant FCRL1, -3 and -5 proteins (R&D Systems). Recombinant FCRL4 protein was not available for this study. Commercial and home-made anti-FCRL polyclonal antibodies were included as positive controls in ELISA experiment as follows: reactivities of recombinant FCRL1 and -5 proteins were confirmed by commercial goat anti-human FCRL polyclonal antibodies (R&D Systems) and reactivity of recombinant FCRL3 was approved by polyclonal rabbit anti-FCRL antibody produced in our laboratory (data not shown). The latter antibody was produced using FCRL peptide and was not reactive by flow cytometry.

Article Snippet: 7G7 refers to a positive control commercial mAb obtained from Genentech Co. (c) ELISA results obtained for anti-FCRL2 mAbs showing specific binding to recombinant FCRL2 protein with no cross-reactivity to eukaryotic recombinant FCRL1, -3 and -5 proteins (R&D Systems).

Techniques: Bioprocessing, Western Blot, Recombinant, Transfection, Marker, Flow Cytometry, Plasmid Preparation, Staining, Negative Control, Positive Control, Enzyme-linked Immunosorbent Assay, Binding Assay, Produced

Journal: Immunology

Article Title: Ligation of human Fc receptor like-2 by monoclonal antibodies down-regulates B-cell receptor-mediated signalling

doi: 10.1111/imm.12311

Figure Lengend Snippet: Influence of anti- Fc receptor like-2 (FCRL2) monoclonal antibodies (mAbs) on calcium mobilization (a) and protein tyrosine phosphorylation profiles (b) induced by anti-IgM antibody in the CA46 Burkitt's lymphoma (BL) cell line. (a) FCRL2-specific mAbs inhibit the calcium mobilization induced by anti-IgM antibody in the CA46 BL cell line. Calcium mobilization was analysed in Fluo-4FF loaded CA46 cells which were treated with saturating amounts of anti-IgM antibody together with (I) 5A7-E7, (II) 3D8-G8 and (III) negative control (anti-Env11, specific to HIV envelope protein, IgG1/k) mAbs. Baseline fluorescence was recorded for 30 seconds at 37° and subsequently the antibodies were added in appropriate tubes and fluorescence was continuously recorded in the FL1 channel by flow cytometry. Black lines represent anti-IgM and grey lines denote anti-IgM and anti-FCRL2 mAbs. The experiments were repeated twice. (b) Protein tyrosine phosphorylation profiles in the CA46 BL cell line stimulated with anti-FCRL2 mAb (5A7-E7) and different concentrations of anti-IgM antibody. The CA46 cell line was treated with different concentrations of anti-BCR antibody and constant concentration of anti-FCRL2 mAb for 1 and 3 min at 37°. Cells were lysed with 1% Triton X-100 and immunoblotted with phospho-tyrosine-specific antibody (clone 4G10). More obvious inhibition of some phosphoproteins was seen especially at 1 min after treatment and also at lower concentrations of anti-IgM antibody.

Article Snippet: 7G7 refers to a positive control commercial mAb obtained from Genentech Co. (c) ELISA results obtained for anti-FCRL2 mAbs showing specific binding to recombinant FCRL2 protein with no cross-reactivity to eukaryotic recombinant FCRL1, -3 and -5 proteins (R&D Systems).

Techniques: Bioprocessing, Phospho-proteomics, Negative Control, Fluorescence, Flow Cytometry, Concentration Assay, Inhibition

Journal: Immunology

Article Title: Ligation of human Fc receptor like-2 by monoclonal antibodies down-regulates B-cell receptor-mediated signalling

doi: 10.1111/imm.12311

Figure Lengend Snippet: Inhibitory influence of anti-Fc receptor like-2 (FCRL2) monoclonal antibody (mAb; 5A7-E7) on phosphorylation status of Erk1/2 and p38 mitogen-activated protein kinase (MAPK) in the CA46 Burkitt's lymphoma (BL) cell line. (a) Stimulation was done with different concentrations of anti-IgM antibody (10, 5 and 2·5 μg/ml) for 1 and 3 min. (b) Stimulation was performed with 5 μg/ml of anti-IgM antibody for 1, 3 and 5 min. The FCRL2-positive CA46 cell line was stimulated with anti-FCRL2 and different concentrations of anti-IgM antibodies for 1, 3 and/or 5 min. The cells were lysed and immunoblotted with phospho-specific anti-p-Erk1/2 and anti-p-p38 mAbs then stripped filters were immunoblotted with anti-β-actin antibodies as loading control. The band density was quantified by laser densitometry and the results are shown relative to β-actin. The error bars represent the standard deviations obtained from three independent experiments. * and ** denote significant inhibition of Erk and p38 phosphorylation induced by anti-FCRL2 mAb relative to anti-IgM antibody alone at different stimulation time intervals (*< 0·05 and **< 0·01).

Article Snippet: 7G7 refers to a positive control commercial mAb obtained from Genentech Co. (c) ELISA results obtained for anti-FCRL2 mAbs showing specific binding to recombinant FCRL2 protein with no cross-reactivity to eukaryotic recombinant FCRL1, -3 and -5 proteins (R&D Systems).

Techniques: Phospho-proteomics, Control, Inhibition

Journal: Immunology

Article Title: Ligation of human Fc receptor like-2 by monoclonal antibodies down-regulates B-cell receptor-mediated signalling

doi: 10.1111/imm.12311

Figure Lengend Snippet: Inhibitory influence of anti- Fc receptor like-2 (FCRL2) monoclonal antibody (mAb; 5A7-E7) on phosphorylation status of B-cell receptor (BCR) signalling mediators in the CA46 Burkitt's lymphoma (BL) cell line. The FCRL2-positive CA46 cell line was stimulated with anti-FCRL2 and anti-IgM antibodies for 1, 3 and 5 min. The cells were lysed and immunoblotted with phospho-specific mAbs then stripped filters were immunoblotted with anti-β-actin antibodies as loading control. The band density was quantified by laser densitometry. (a) Immunoblotting with anti-p-Vav and anti-p-Akt and densitometry results related to p-Vav/β-actin and Akt/β-actin indicated that BCR-induced phosphorylation of Akt was not significantly influenced and Vav phosphorylation was slightly reduced by FCRL2 ligation. The phosphorylation status of Syk (b), Jnk1 (c) and IKK (d) in the CA46 BL cell line stimulated with anti-IgM antibody and anti-FCRL2 mAb (5A7-E7) showed that Syk and Jnk1 phosphorylation was inhibited by FCRL2 signalling, however, IKK was not affected by FCRL2 stimulation. The experiments were performed twice.

Article Snippet: 7G7 refers to a positive control commercial mAb obtained from Genentech Co. (c) ELISA results obtained for anti-FCRL2 mAbs showing specific binding to recombinant FCRL2 protein with no cross-reactivity to eukaryotic recombinant FCRL1, -3 and -5 proteins (R&D Systems).

Techniques: Phospho-proteomics, Control, Western Blot, Ligation

Journal: Immunology

Article Title: Ligation of human Fc receptor like-2 by monoclonal antibodies down-regulates B-cell receptor-mediated signalling

doi: 10.1111/imm.12311

Figure Lengend Snippet: Anti- Fc receptor like-2 (FCRL2) monoclonal antibody (mAb; 5A7-E7) has no inhibitory influence on the phosphorylation status of whole protein tyrosine (a), Erk (b) and p38 (c) in the anti-IgM antibody stimulated DG75 cell line. The FCRL2-negative DG75 cell line was treated with anti-BCR antibody and anti-FCRL2 mAb for 1, 3 and 5 min at 37°. Cells were lysed with 1% Triton X-100 and immunoblotted with phospho-tyrosine-specific (clone 4G10), p-Erk1/2 and p-p38 antibodies. Stripped filters were immunoblotted with anti-β-actin antibodies as loading control and the band density was quantified by laser densitometry. These experiments were done as control in parallel under the same conditions adapted for the CA46-treated cell line. Phosphorylation of whole tyrosine protein, p-Erk1/2 and p-p38 was not altered by anti-FCRL2 mAb. Phosphorylation of Erk1/2 was assessed at 5 μg/ml of anti-IgM antibody and phosphorylation of whole tyrosine protein and p38 was checked at 10 μg/ml of anti-IgM antibody.

Article Snippet: 7G7 refers to a positive control commercial mAb obtained from Genentech Co. (c) ELISA results obtained for anti-FCRL2 mAbs showing specific binding to recombinant FCRL2 protein with no cross-reactivity to eukaryotic recombinant FCRL1, -3 and -5 proteins (R&D Systems).

Techniques: Phospho-proteomics, Control