Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: RETRACTED: Blockade of TNF-α signaling suppresses the AREG-mediated IL-6 and IL-8 cytokines secretion induced by anti-Ro/SSA autoantibodies.

doi: 10.1038/labinvest.2010.168

Figure Lengend Snippet: Figure 1 Semiquantitative RT-PCR and real-time PCR for Furin, TACE and AREG genes expression. (a) RT-PCR analysis of Furin, TACE and AREG mRNA extracted from SGEC treated with anti-Ro/SSA autoantibodies. M, marker; control, untreated SGEC; HIgG, SGEC treated with IgG fractions extracted from sera of healthy donors; anti-Ro, SGEC treated with anti-Ro/SSA autoantibodies. RT-PCR of GADPH was used as control. Band intensities were analyzed by densitometry (b). (c) Real-time PCR for Furin, TACE and AREG genes expression. Representative histograms of mRNA levels of Furin, TACE and AREG in untreated SGEC (control), SGEC treated with healthy IgG (HIgG), anti-Ro/SSA autoantibodies (anti-Ro). The mRNA levels of the housekeeping gene, b-2 microglobulin, were quantified between untreated control cells and variously treated cells (data represent the mean±s.e. of five independent experiments).

Article Snippet: Membranes were incubated for 90 min with rabbit anti-human Furin polyclonal antibody (pAb), goat antihuman TACE pAb (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human AREG mAb (R&D Systems, Minneapolis, MN USA), and for 30 min with the relative secondary antibodies-HRP conjugates (Santa Cruz Biotechnology).

Techniques: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing, Marker, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: RETRACTED: Blockade of TNF-α signaling suppresses the AREG-mediated IL-6 and IL-8 cytokines secretion induced by anti-Ro/SSA autoantibodies.

doi: 10.1038/labinvest.2010.168

Figure Lengend Snippet: Figure 2 Analysis of Furin, TACE and AREG expression in anti-Ro/SSA Abs-treated SGEC. (a) Flow cytometric analysis of Furin, TACE and AREG expression in SGEC after anti-Ro/SSA Abs treatment. Examples of flow cytometric images from one representative experiment. (A) Furin expression analysis in untreated and anti-Ro/SSA or HIgG-treated SGEC; (B) intracellular active TACE expression analysis in untreated and anti-Ro/SSA or HIgG-treated SGEC; (C) AREG expression analysis in untreated and anti-Ro/SSA or HIgG-treated SGEC. (b) Western blot analysis of Furin, TACE and AREG proteins expression in SGEC treated or not with anti-Ro/SSA. Immunoblotting gave rise to bands of the expected size (97 kDa for Furin, 80 kDa for active TACE and 50 kDa for AREG). b-Actin was used as protein loading control. (c) Detection of soluble AREG by ELISA. Secreted AREG was detected by ELISA in the conditioned medium. Control, untreated SGEC; HIgG, SGEC treated with IgG fractions extracted from sera of healthy donors; anti-Ro, SGEC treated with anti-Ro/SSA autoantibodies. (Data represent the mean±s.e. of four independent experiments).

Article Snippet: Membranes were incubated for 90 min with rabbit anti-human Furin polyclonal antibody (pAb), goat antihuman TACE pAb (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human AREG mAb (R&D Systems, Minneapolis, MN USA), and for 30 min with the relative secondary antibodies-HRP conjugates (Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2

doi: 10.3390/ijms23169306

Figure Lengend Snippet: TNF-α does not impact the levels of TNFR1 and TNFR2 on Th1 and Th17 cells. Purified CD4 + T cell subsets were stimulated with 1 µg/mL of TNF-α for 24 h and stained, for flow cytometry, with anti-human TNFR1 or TNFR2 mAbs. ( A ) Representative histograms showing the expression levels of TNFR1 and TNFR2 on Th1 and Th17 cells at baseline conditions and upon TNF-α stimulus. Solid black line histograms, isotype control; black histograms, Th1 cells; grey histograms, Th17 cells; dashed line histograms, TNF-α-treated Th1 cells; long dashed line histograms, TNF-α-treated Th17 cells. The levels of TNFR1 ( B ) and TNFR2 ( C ) were measured on purified CD4 + T cell subpopulations obtained from two to four healthy controls. Bars represent the mean values ± SD. Statistical analyses were carried out with Kruskal–Wallis followed by Dunn’s multiple comparison tests. ** p < 0.01.

Article Snippet: Cells were then stained with a mouse anti-human TNFR1-APC mAb (clone 16803) (R&D Systems, Minneapolis, MN, USA) and a rat anti-human TNFR2-PE mAb (clone hTNFR-M1) (BD Biosciences) for 30 min at 4 °C in the dark.

Techniques: Purification, Staining, Flow Cytometry, Expressing, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2

doi: 10.3390/ijms23169306

Figure Lengend Snippet: Effect of TNFR1 or TNFR2 blockade on the production of IFN-γ and IL-17 by Th1 and Th17 cells. Purified CD4 + T lymphocyte subpopulations were incubated with neutralizing antibodies to TNFR1 or TNFR2 for 1 h prior to a 4-day stimulus with TNF-α (1 µg/mL). Intracellular staining of IFN-γ and IL-17 was assessed by flow cytometry. ( A ) Representative dot plots of Th1 and Th17 cells treated with anti-TNFR1 or anti-TNFR2 in the presence or absence of TNF-α. An isotype control was used to discard non-specific effects of the neutralizing antibodies. The fold increase in the percentages of IFN-γ ( B , D ) or IL-17 ( C , E ) producers was measured on Th1 and Th17 cells obtained from two to six healthy donors. Bars represent the mean values ± SD. Data were normalized against cells that did not receive treatment with TNF-α or TNF-α plus TNFRs blocking mAbs. For statistical analysis, Kruskal–Wallis and Dunn’s multiple comparison tests were performed. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cells were then stained with a mouse anti-human TNFR1-APC mAb (clone 16803) (R&D Systems, Minneapolis, MN, USA) and a rat anti-human TNFR2-PE mAb (clone hTNFR-M1) (BD Biosciences) for 30 min at 4 °C in the dark.

Techniques: Purification, Incubation, Staining, Flow Cytometry, Control, Blocking Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2

doi: 10.3390/ijms23169306

Figure Lengend Snippet: Expression of TNFR1 and TNFR2 on Th1 and Th17 cells present in the peripheral blood of rheumatoid arthritis (RA) patients treated with adalimumab. Cell staining for flow cytometry analysis was performed on PBMC samples from healthy controls ( n = 9) and RA patients ( n = 10) before (PRE) and after (POST) treatment with adalimumab. The levels of TNFR1 ( A ) and TNFR2 ( C ) are expressed in MFI values. The frequency of TNFR1 ( B ) and TNFR2 ( D )-expressing lymphocytes are also shown. Each symbol represents data for one individual. Mean values ± SD are indicated. Significance was assessed with non-parametric Kruskal–Wallis test followed by Dunn’s multiple comparison test (for MFI data) or parametric one-way ANOVA plus Tukey’s post-test (for lymphocyte frequencies data). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cells were then stained with a mouse anti-human TNFR1-APC mAb (clone 16803) (R&D Systems, Minneapolis, MN, USA) and a rat anti-human TNFR2-PE mAb (clone hTNFR-M1) (BD Biosciences) for 30 min at 4 °C in the dark.

Techniques: Expressing, Staining, Flow Cytometry, Comparison

Journal:

Article Title: The Cysteine-Rich Region of the Entamoeba histolytica Adherence Lectin (170-Kilodalton Subunit) Is Sufficient for High-Affinity Gal/GalNAc-Specific Binding In Vitro

doi:

Figure Lengend Snippet: Immunoreactivity of 3F4 with the native lectin heavy subunit. (a) Indirect immunofluorescence of HM1:IMSS trophozoites by flow cytometric analysis. Primary antilectin MAbs 1G7, 3F4, and 8A3 (CLB was the isotype control used to measure background) were monitored by secondary goat anti-mouse-fluorescein isothiocyanate. Data depict fluorescence intensity on a logarithmic scale for 104 trophozites from a representative experiment. (b) Immunoprecipitation of biotinylated native lectin heavy subunit (170 kDa) from solubilized amebic membranes, using antilectin MAb 3F4 (IgG1) and irrelevant isotype control CLB (IgG1). Immunoprecipitates were subject to SDS-PAGE on a 7.5% gel, transferred to nitrocellulose, and probed with avidin-horseradish peroxidase.

Article Snippet: Cells were washed three times with PBS and incubated with a 1:100 dilution of goat anti-mouse-fluorescein isothiocyanate in 0.5% BSA–PBS (Bio-Rad) for 30 min on ice.

Techniques: Immunofluorescence, Fluorescence, Immunoprecipitation, SDS Page, Avidin-Biotin Assay

Journal: Immunity

Article Title: Analysis of a Therapeutic Antibody Cocktail Reveals Determinants for Cooperative and Broad Ebolavirus Neutralization

doi: 10.1016/j.immuni.2020.01.001

Figure Lengend Snippet:

Article Snippet: ELISA plates were coated overnight at 4°C with 0.1 μg/mL of mouse anti-human IgG (human CH2 domain with no cross-reactivity to rhesus macaque IgG; clone R10Z8E9; BioRad) and then blocked for 2 h. The serum samples were assayed at 4-fold dilutions starting at a 1:100 dilution in ELISA diluent (PBS containing 1% heat-inactivated FBS and 0.2% Tween-20).

Techniques: Recombinant, Virus, Mutagenesis, Expressing, Diagnostic Assay, Staining, Plasmid Preparation, Software, Flow Cytometry, Electro Cell Manipulation, Chromatography, Microscopy

Journal: Scientific Reports

Article Title: Characterization of an anti-fetal AChR monoclonal antibody isolated from a myasthenia gravis patient

doi: 10.1038/s41598-017-14350-8

Figure Lengend Snippet: Overview of IgG1-131 & IgG4-131 cloning strategy and production. The expression cassettes for the production of (a) VH-CH and (b) Vκ-Cκ segments of the mAb 131 in IgG format are shown. (c) Isotype specificities of the recombinant antibodies along with control mAbs were analyzed by dot blot using isotype specific HRP-conjugated antibodies.

Article Snippet: Purified mAbs IgG1-131, IgG4-131, as well as mAbs IgG1-637 & IgG4-637 as controls, were blotted on nitrocellulose membranes (Biorad, Cat No. 162-0116) for 1 h at 37 °C, blocked with 5% non-fat dry milk in 1x PBS for 1 h at 37 °C, washed with PBS-T (containing 0.05% Tween20) and incubated with HRP-conjugated goat F(ab’) 2 anti-human IgG-Fcγ (Jackson Immuno-Research, Cat No. 109-036-008; diluted 1:2500) or mouse anti-human IgG1 (Invitrogen, Cat No. 05-3320; diluted 1:2500) or mouse anti-human IgG4 (AbD serotec, Cat No. MCA2098P; diluted 1:2500) secondary antibody.

Techniques: Cloning, Expressing, Recombinant, Control, Dot Blot

Journal: Scientific Reports

Article Title: Characterization of an anti-fetal AChR monoclonal antibody isolated from a myasthenia gravis patient

doi: 10.1038/s41598-017-14350-8

Figure Lengend Snippet: Recombinant IgG1-131 binds fetal AChR and γ-subunit with high specificity and apparent affinity. (a) The α, β and γ-subunits of the human muscle AChR were resolved on SDS-PAGE (shown in upper panel) and specificity of IgG1-131 for γ - subunit was analyzed by Western blot (lower panel). Full image of the SDS-PAGE and Western blot are shown in Supplementary Fig. . (b) ELISA analysis of IgG1-131 (0.01 nM – 10 nM) binding to the α, β and γ-subunit of the AChR. (c) Radio-immunoprecipitation of IgG1-131 using fetal and adult AChR extracted from TE671 and CN21 cell membranes respectively. (d) Cross-reactivity of IgG1-131 against rat and mouse muscle AChR, and the Torpedo electric organ AChR was analyzed by radio-immunoprecipitation assay. (e) The specificities of IgG1-131 and IgG4-131 were tested against fetal (TE671 membrane extract) and adult AChR (CN21 membrane extract). IgG1-637 and IgG4-637 were used as a reference in both tests. (f) 125 I-α-BT blocking was measured as percentage inhibition in the 125 I-α-BT binding to AChRs after co-incubating the individual antibody/AMC serum and 125 I-α-BT labelled AChR. The results are from two independent experiments. (g) Analysis of competition between IgG1-131 and IgG1-637. IgG1-131 (100 nM) when co-incubated with IgG1-637 (25 nM), reduced the amount of 125 I-α-BT co-precipitated with fetal AChR compared to 25 nM IgG1-637 alone. (h) Schematic representation of the binding effects of mAb 131 and mAb 637. The mAb 637 binds to the two designated α-subunits of the fetal AChR, allowing simultaneous binding of two α-BT molecules. However, in the presence of competing/blocking mAb 131, one of the α-BT molecules and mAb 637 at the α/γ interface gets displaced by mAb 131.

Article Snippet: Purified mAbs IgG1-131, IgG4-131, as well as mAbs IgG1-637 & IgG4-637 as controls, were blotted on nitrocellulose membranes (Biorad, Cat No. 162-0116) for 1 h at 37 °C, blocked with 5% non-fat dry milk in 1x PBS for 1 h at 37 °C, washed with PBS-T (containing 0.05% Tween20) and incubated with HRP-conjugated goat F(ab’) 2 anti-human IgG-Fcγ (Jackson Immuno-Research, Cat No. 109-036-008; diluted 1:2500) or mouse anti-human IgG1 (Invitrogen, Cat No. 05-3320; diluted 1:2500) or mouse anti-human IgG4 (AbD serotec, Cat No. MCA2098P; diluted 1:2500) secondary antibody.

Techniques: Recombinant, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay, Radio Immunoprecipitation, Membrane, Blocking Assay, Inhibition, Incubation

Journal: Scientific Reports

Article Title: Characterization of an anti-fetal AChR monoclonal antibody isolated from a myasthenia gravis patient

doi: 10.1038/s41598-017-14350-8

Figure Lengend Snippet: IgG1-131 binds to human RMS cell lines. (a) Binding of IgG1-131 and IgG1-637 to cell surface fetal AChR on different RMS cell lines was analyzed by immunofluorescence. Binding of antibody was detected by goat anti-human IgG-Alexa 488 antibody (green) and Hoechst was used to stain the nuclei. HEK293 cells were used as negative control for each antibody. Representative images at 20x magnification are shown, n = 2. Scale bar = 50 µm. (b) Analysis of 125 I-α-BT bound to cell surface AChR on RMS cell lines after incubation with IgG1-131 or IgG1 637. Individual bars correspond to the mean ± SD of three replicates tested for each condition. Results are representative of 3 independent experiments.

Article Snippet: Purified mAbs IgG1-131, IgG4-131, as well as mAbs IgG1-637 & IgG4-637 as controls, were blotted on nitrocellulose membranes (Biorad, Cat No. 162-0116) for 1 h at 37 °C, blocked with 5% non-fat dry milk in 1x PBS for 1 h at 37 °C, washed with PBS-T (containing 0.05% Tween20) and incubated with HRP-conjugated goat F(ab’) 2 anti-human IgG-Fcγ (Jackson Immuno-Research, Cat No. 109-036-008; diluted 1:2500) or mouse anti-human IgG1 (Invitrogen, Cat No. 05-3320; diluted 1:2500) or mouse anti-human IgG4 (AbD serotec, Cat No. MCA2098P; diluted 1:2500) secondary antibody.

Techniques: Binding Assay, Immunofluorescence, Staining, Negative Control, Incubation

Journal: Scientific Reports

Article Title: Characterization of an anti-fetal AChR monoclonal antibody isolated from a myasthenia gravis patient

doi: 10.1038/s41598-017-14350-8

Figure Lengend Snippet: IgG1-131 does not lead to modulation of fetal AChR on TE671 cells, but partly displaces 125 I-α-BT. The effect of (a) IgG1-637, (b) IgG4-637, (c) IgG1-131 and (d) IgG4-131 on the 125 I-α-BT binding to TE671 (fetal AChR) and CN21 (adult AChR) cells is shown as normalized cell bound radioactivity at varying molar concentrations of the antibodies. Cells were incubated with antibodies for 3 h and subsequently labelled with 125 I-α-BT. Each data point corresponds to the mean ± SD of three replicates. (e) Effect of IgG1-131 and IgG1-637 on 125 I-α-BT binding to TE671 cells at different time points (1, 2 and 3 h). For each time point, cells cultured in medium without any IgG were used as controls. Unspecific binding of 125 I-α-BT was measured by pre-incubation of cells with an excess of unlabeled α-BT (25 nM). Data points of 1, 2 and 3 hours incubation were pooled for this condition. (f) Schematic representation of the Fab arm-exchange reaction under reducing conditions between IgG4-131 (VH and VL domains shown in red and CH in black color) and IgG4 from normal human serum (IgG4-NHS; VH and VL domains shown in blue and CH in grey color) to yield two bispecific IgG4 molecules. (g) Purity of IgG4 isolated from normal human serum (NHS) was validated by dot blot assay along with control mAbs. (h) Competition experiments using monovalent FAE IgG4-131 and IgG4-637. The loss of the cell bound radioactivity due to the IgG1-131 incubation was not significantly affected by an excess FAE IgG4-131. In contrast, FAE IgG4-637 completely counteracted the effect of IgG1-637. Data are pooled from 3 experiments with representative groups.

Article Snippet: Purified mAbs IgG1-131, IgG4-131, as well as mAbs IgG1-637 & IgG4-637 as controls, were blotted on nitrocellulose membranes (Biorad, Cat No. 162-0116) for 1 h at 37 °C, blocked with 5% non-fat dry milk in 1x PBS for 1 h at 37 °C, washed with PBS-T (containing 0.05% Tween20) and incubated with HRP-conjugated goat F(ab’) 2 anti-human IgG-Fcγ (Jackson Immuno-Research, Cat No. 109-036-008; diluted 1:2500) or mouse anti-human IgG1 (Invitrogen, Cat No. 05-3320; diluted 1:2500) or mouse anti-human IgG4 (AbD serotec, Cat No. MCA2098P; diluted 1:2500) secondary antibody.

Techniques: Binding Assay, Radioactivity, Incubation, Cell Culture, Isolation, Dot Blot, Control

Journal: Scientific Reports

Article Title: Characterization of an anti-fetal AChR monoclonal antibody isolated from a myasthenia gravis patient

doi: 10.1038/s41598-017-14350-8

Figure Lengend Snippet: IgG1-131 does not inhibit fetal AChR function. (a) AChR currents (mean ± SEM) in individual cells (n = 6) were measured during the continuous perfusion with control solution (without IgG1-131) and with test solution (application of 5 μg/mL purified IgG1-131 for 2.5 min). (b) Repetitive AChR current measurements in an individual cell continuously perfused with 10 mL of test solution. Data points are the means ( ± SEM) of a number of measurements (control solution: n = 10, test solution: n = 6 at 0.5 min, n = 6 at 8 min, n = 6 at 15 min and n = 3 at 22 min). AChR currents were normalized to the cell capacitance and expressed as pA/pF.

Article Snippet: Purified mAbs IgG1-131, IgG4-131, as well as mAbs IgG1-637 & IgG4-637 as controls, were blotted on nitrocellulose membranes (Biorad, Cat No. 162-0116) for 1 h at 37 °C, blocked with 5% non-fat dry milk in 1x PBS for 1 h at 37 °C, washed with PBS-T (containing 0.05% Tween20) and incubated with HRP-conjugated goat F(ab’) 2 anti-human IgG-Fcγ (Jackson Immuno-Research, Cat No. 109-036-008; diluted 1:2500) or mouse anti-human IgG1 (Invitrogen, Cat No. 05-3320; diluted 1:2500) or mouse anti-human IgG4 (AbD serotec, Cat No. MCA2098P; diluted 1:2500) secondary antibody.

Techniques: Control, Purification