Journal: Cell Reports Medicine

Article Title: MAIT cell activation is associated with disease severity markers in acute hantavirus infection

doi: 10.1016/j.xcrm.2021.100220

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-IL-12 (p40/p70) , Milteny Biotec , Cat#130-095-755, clone C8.6.

Techniques: Control, Virus, Clinical Proteomics, Recombinant, Staining, Luminex, Enzyme-linked Immunosorbent Assay, Labeling, Software

Journal: The Journal of Biological Chemistry

Article Title: Deciphering site 3 interactions of interleukin 12 and interleukin 23 with their cognate murine and human receptors

doi: 10.1074/jbc.RA120.013935

Figure Lengend Snippet: IL-23p19W157 and IL-12p35Y185 in mouse are functional hot-spot amino acids. A, structural superpositioning of crystal structures of human IL-23p19 and IL-12p35 (PDB codes 3D87 and 3HMX) with models of their murine counterparts. B, cellular proliferation of Ba/F3-gp130 cells with cDNAs coding for mIL-23R and mIL-12Rβ1. The cells were cultured for 3 days in the presence of 10 ng/ml HIL-6 or with the indicated cytokines (10% conditioned cell culture supernatant). Parental Ba/F3-gp130 cells were used as controls. The results of one representative experiment of five are shown. Error bars represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). C, Western blotting (WB) of secreted mp40–p19 and mp40–p19W157A from transfected CHO-K1 cells. D, analysis of STAT3 and ERK1/2 activation. Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells were washed, starved, and stimulated with the indicated cytokines (10% conditioned cell culture supernatant) for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 µg/lane) were loaded on SDS-PAA gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-ERK1/2, and ERK. Western blotting data show results of one representative experiment of four. E, co-IP of FLAG-tagged mp40–p35 and mp40–p35Y185R by Fc-tagged smIL-12Rβ1 or smIL-12Rβ2. Lanes L, lysates; lanes +, coimmunoprecipitates; lanes C, controls (without smIL-12Rβ1-Fc or smIL-12Rβ2-Fc). One of two independent experiments is shown. F, cellular proliferation of Ba/F3-gp130 cells with cDNAs coding for mIL-12Rβ2 and mIL-12Rβ1. The assay was performed as described for B. The results of one representative experiment of four are shown. Error bars represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). G, Western blotting of secreted mp40–p35 and mp40–p35Y185R from transfected CHO-K1 cells. H, analysis of STAT3 and ERK1/2 activation. The assay was performed with BaF3-gp130-mIL-12Rβ1-mIL-12Rβ2 cells as described for D. Western blotting data show the results of one representative experiment of two. EGFP, enhanced green fluorescent protein; ns, not significant; RFU, relative fluorescence units. ***, P ≤ 0.001.

Article Snippet: Biotinylated hIL-23R (BAF1400), hIL-12Rβ1 (BAF839), hIL-12Rβ2 (BAF1959), mp40 (BAF499), and hp40 (BAF219) mAbs and hIL-12Rβ1 phycoerythrin-conjugated mAb (FAB839P) were from R&D Systems (Minneapolis, MN).

Techniques: Functional Assay, Cell Culture, Western Blot, Transfection, Activation Assay, Co-Immunoprecipitation Assay, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Deciphering site 3 interactions of interleukin 12 and interleukin 23 with their cognate murine and human receptors

doi: 10.1074/jbc.RA120.013935

Figure Lengend Snippet: Replacement of W156 by glutamic acid did not influence activity of hp40–p19 on Ba/F3 cells with human receptors. A, cellular proliferation of Ba/F3-gp130 cells with cDNAs coding for mIL-23R and mIL-12Rβ1. The cells were cultured for 3 days in the presence of 10 ng/ml HIL-6 or with the indicated cytokines (10% conditioned cell culture supernatant). Parental Ba/F3-gp130 cells were used as controls. The results of one representative experiment of two are shown. Error bars represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). B, analysis of STAT3 and ERK1/2 activation. Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells were washed, starved, and stimulated with the indicated cytokines (10% conditioned cell culture supernatant) for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 µg/lane) were loaded on SDS-PAA gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-ERK1/2, and ERK. Western blotting data show results of one representative experiment of two. C, cellular proliferation of Ba/F3 cells with cDNAs coding for hIL-23R and hIL-12Rβ1. The cells were cultured for 3 days in the presence of IL-3 (2% conditioned cell culture supernatant of WEHI-3B cells) or with the indicated cytokines (10% conditioned cell culture supernatant). Parental Ba/F3 cells were used as controls. The results of one representative experiment of two are shown. Error bars represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). D, analysis of STAT3 and ERK1/2 activation. The assay was performed with Ba/F3-hIL-12Rβ1-hIL-23R cells as described for B. Western blotting data show results of one representative experiment of two. E, Western blotting of secreted hp40–p19, hp40–p19W156A, and hp40–p19W156E from transfected CHO-K1 cells. Cytokine variants were transiently expressed with comparable efficiency. F, cellular proliferation of Ba/F3 cells with cDNAs coding for hIL-23R and hIL-12Rβ1. The assay was performed as described in C. The results of one representative experiment of two are shown. Error bars represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). G, analysis of STAT3 and ERK1/2 activation. The assay was performed with Ba/F3-hIL-12Rβ1-hIL-23R cells as described for B. Western blotting data show results of one representative experiment of two. H, cellular proliferation of Ba/F3-gp130 cells with cDNAs coding for mIL-23R and mIL-12Rβ1. The assay was performed as described for A. The results of one representative experiment of two are shown. Error bars represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). I, analysis of STAT3 and ERK1/2 activation. The assay was performed with Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells as described for B. Western blotting data show results of one representative experiment of two. J, Western blotting of secreted mp40–p19, mp40–p19W157A and mp40–p19W157E from transfected CHO-K1 cells. Cytokine variants were transiently expressed with comparable efficiency. EGFP, enhanced green fluorescent protein; ns, not significant; RFU, relative fluorescence units. ***, P ≤ 0.001.

Article Snippet: Biotinylated hIL-23R (BAF1400), hIL-12Rβ1 (BAF839), hIL-12Rβ2 (BAF1959), mp40 (BAF499), and hp40 (BAF219) mAbs and hIL-12Rβ1 phycoerythrin-conjugated mAb (FAB839P) were from R&D Systems (Minneapolis, MN).

Techniques: Activity Assay, Cell Culture, Activation Assay, Western Blot, Transfection, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Deciphering site 3 interactions of interleukin 12 and interleukin 23 with their cognate murine and human receptors

doi: 10.1074/jbc.RA120.013935

Figure Lengend Snippet: Murine IL-23 with human site 3 substitution is still active on Ba/F3 cells with human receptors. A, alignment of murine and human IL-23p19 site 3 amino acids. B, Western blotting of secreted mp40–p19 variants from transfected CHO-K1 cells. Cytokine variants were transiently expressed with comparable efficiency. C, cellular proliferation of Ba/F3-gp130 cells with cDNAs coding for mIL-23R and mIL-12Rβ1. The cells were cultured for 3 days in the presence of 10 ng/ml HIL-6 or with the indicated cytokines (10% conditioned cell culture supernatant). Parental Ba/F3-gp130 cells were used as controls. The results of one representative experiment of five are shown. Error bars represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). D, analysis of STAT3 and ERK1/2 activation. Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells were washed, starved, and stimulated with the indicated cytokines (10% conditioned cell culture supernatant) for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 µg/lane) were loaded on SDS-PAA gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-ERK1/2, and ERK. Western blotting data show results of one representative experiment of four. E, cellular proliferation of Ba/F3 cells with cDNAs coding for hIL-23R and hIL-12Rβ1. The cells were cultured for 3 days in the presence of IL-3 (2% conditioned cell culture supernatant of WEHI-3B cells) or with the indicated cytokines (10% conditioned cell culture supernatant). Parental Ba/F3 cells were used as controls. The results of one representative experiment of five are shown. Error bars represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). F, analysis of STAT3 and ERK1/2 activation. The assay was performed with Ba/F3-hIL-12Rβ1-hIL-23R cells as described for D. Western blotting data show results of one representative experiment of four. EGFP, enhanced green fluorescent protein; ns, not significant; RFU, relative fluorescence units. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Article Snippet: Biotinylated hIL-23R (BAF1400), hIL-12Rβ1 (BAF839), hIL-12Rβ2 (BAF1959), mp40 (BAF499), and hp40 (BAF219) mAbs and hIL-12Rβ1 phycoerythrin-conjugated mAb (FAB839P) were from R&D Systems (Minneapolis, MN).

Techniques: Western Blot, Transfection, Cell Culture, Activation Assay, Fluorescence

Journal:

Article Title: Human IL-23-producing type 1 macrophages promote but IL-10-producing type 2 macrophages subvert immunity to (myco)bacteria

doi: 10.1073/pnas.0400983101

Figure Lengend Snippet: Differentiation of human blood monocytes into functionally distinct Mϕ subsets, Mϕ-1 and Mϕ-2. (A) In contrast to mo.DCs, Mϕ-1 and Mϕ-2 highly expressed CD14 but showed no or only weak expression of CD1a or CD83 (the latter after activation by LPS) as determined by flow cytometry. Cytokine secretion was measured up to 72 h after stimulation with M. tuberculosis sonicate (myc) (▪), LPS (•), or medium control (♦). (B) Whereas activated Mϕ-1, similar to DCs, secreted high levels of IL-12p40, Mϕ-2 failed to secrete IL-12p40. (C) IL-10, in contrast, was most predominant in Mϕ-2. Similar cytokine profiles were obtained with cells from at least 10 independent donors.

Article Snippet: IL-23 was measured by ELISA using anti-IL-12p40 monoclonal antibody BP40 (Diaclone) for coating and rat-anti-hp19 monoclonal antibody 12F12 for detection (with a sensitivity of 60 pg/ml).

Techniques: Expressing, Activation Assay, Flow Cytometry

Journal:

Article Title: Human IL-23-producing type 1 macrophages promote but IL-10-producing type 2 macrophages subvert immunity to (myco)bacteria

doi: 10.1073/pnas.0400983101

Figure Lengend Snippet: Type 1 cytokine secretion by monocyte-derived Mϕ and mo.DCs. The capacity of mo.DCs, Mϕ-1, and Mϕ-2 to secrete IL-12 (p40/p35) and IL-23 (p40/p19) proteins was determined 24 h after microbial stimulation in the absence (black bars) or presence (gray bars) of 500 units/ml IFN-γ. Mϕ-1 secreted IL-23 but failed to produce IL-12 after activation with mycobacterial sonicate (10 μg/ml) or LPS (10 ng/ml) unless IFN-γ was added. Also, DCs showed IFN-γ-enhanced IL-12 secretion, but unlike Mϕ-1 they yielded decreased rather than elevated IL-23 levels with IFN-γ. Mϕ-2 failed to produce IL-12 or IL-23. Depicted are average protein levels plus standard deviation (n = 3; n = 5 for IL-12 in DCs and Mϕ-1).

Article Snippet: IL-23 was measured by ELISA using anti-IL-12p40 monoclonal antibody BP40 (Diaclone) for coating and rat-anti-hp19 monoclonal antibody 12F12 for detection (with a sensitivity of 60 pg/ml).

Techniques: Derivative Assay, Activation Assay, Standard Deviation

Journal:

Article Title: Human IL-23-producing type 1 macrophages promote but IL-10-producing type 2 macrophages subvert immunity to (myco)bacteria

doi: 10.1073/pnas.0400983101

Figure Lengend Snippet: Type 1 cytokine mRNA levels in monocyte-derived Mϕ and mo.DCs. (A) Quantitative reverse transcription–PCR analyses 8 h after LPS stimulation of Mϕ-1 revealed that expression of IL-12-specific p35, but not p40 or p19 mRNA, required costimulation with IFN-γ (500 units/ml). DCs showed low but significant transcription of p35 in response to LPS and profound levels of p40 and p19 mRNA. Mϕ-2 failed to induce any of these cytokine transcripts. (B) Whereas stimulation with 10 μg/ml mycobacterial sonicate strongly induced the production of p40 and p19 mRNA, high-level transcription of p35 required costimulation with IFN-γ. (Note that the y axes are logarithmic in B!) Stimulation indexes were calculated from the average level of mRNA from two (DCs) to four (Mϕ-1) experiments. Activation-induced p19 transcription was inhibited by IFN-γ in both mo.DCs and Mϕ-1.

Article Snippet: IL-23 was measured by ELISA using anti-IL-12p40 monoclonal antibody BP40 (Diaclone) for coating and rat-anti-hp19 monoclonal antibody 12F12 for detection (with a sensitivity of 60 pg/ml).

Techniques: Derivative Assay, Expressing, Activation Assay

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

Article Title: IL-23 is increased in dendritic cells in multiple sclerosis and down-regulation of IL-23 by antisense oligos increases dendritic cell IL-10 production.

doi: 10.4049/jimmunol.176.12.7768

Figure Lengend Snippet: FIGURE 1. IL-23 ELISA calibrating curve. The IL-23 ELISA uses a standard approach. IL-23R/Fc Chimera is used as a capture reagent and anti-human IL-12/IL-23 p40 mAb as a detection Ab (R&D Systems). The sensitivity of the IL-23 ELISA was 78 pg/ml.

Article Snippet: For detecting Ab, 2 g/ml mouse anti-human p40 Ab (clone no. 169516; R&D Systems) was added for 2 h, followed by biotinylated rat anti-mouse mAb (Zymed Laboratories) for an additional 1 h. The sensitivity of the IL-23 ELISA was 78 pg/ml.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Interferon-α and Interleukin-12 Are Induced Differentially by Toll-like Receptor 7 Ligands in Human Blood Dendritic Cell Subsets

doi: 10.1084/jem.20020207

Figure Lengend Snippet: TLR-7 ligands induce the production of IL-12 and IFN-α from MDCs and PDCs, respectively. (A and B) After 24-h culture with various stimuli, concentration of IL-12 p40+p70 (A) and IFN-α (B) in the culture supernatants of MDCs, PDCs, and PBMCs were measured by ELISA. The data are shown by means ± SEM of five independent experiments. (C) After 5-h culture with R-848 (10 −6 M), intracellular staining of IL-12 and IFN-α in MDCs and PDCs, together with the staining of surface expression of CD40, was performed. Percentages of the respective cytokine producing DCs are indicated. This figure represents the results from one of three experiments.

Article Snippet: 10 μg/ml brefeldin A (Sigma-Aldrich) was added during the last 1 h. After the stimulation, MDCs and PDCs were stained with Cy-Chrome-labeled CD40 (5C3; BD PharMingen), and then fixed, permeabilized (FIX and PERM kit; Caltag Laboratories), and stained with FITC-labeled anti–IL-12 p40+p70 mAb (B-P24; DIACLONE Research) or unconjugated mouse anti–human IFN-α mAb (MC-16; Genzyme).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Expressing