ifnar1 neutralizing antibody mar1  (MedChemExpress)

Journal: Inflammation

Article Title: IRF7 Modulates Inflammatory Pain Through Upregulating IFNβ in Mice Trigeminal Ganglion

doi: 10.1007/s10753-026-02483-w

Figure Lengend Snippet: Distribution of IRF7-IFNβ signaling in the wild-type TG. ( A , C , E ) Representative images and quantitative co-localization analysis of IRF7, IFNβ or IFNAR co-labeled with neuronal (NeuN), macrophage (CD68), and SGC (GS) markers in the TG (A,C scale bar = 100 μm; E, scale bar = 50 μm).( B , D ) Immunofluorescence images and quantitative co-localization analysis of IRF7 or IFNβ co-labeled with neuronal subtypes expressing CGRP, P2X3R, or NF in the TG (scale bar = 100 μm). ( F ) t-SNE plots showing the distribution of Irf7, Ifnar1, Ifnar2 and the markers of neuronal subtypes in TG neurons, including Calca (encode CGRP), P2rx3 (encode P2X3R) and Nefh (encode NF), based on single-cell sequencing. ( G ) Proportion of Irf7-positive cells co-expressing Nefh, Calca and P2rx3. ( H ) The distribution of Ifnar1 and Ifnar2 in various TG cell types

Article Snippet: The IFNAR1 neutralizing antibody MAR1 or mouse IgG (HY- P99137 and HY-P99977 purchased from MedChem Express LLC) was delivered intra-ganglionically on 3 dpi, at a dose of 1 μL(10 μg/1 μL).

Techniques: Labeling, Immunofluorescence, Expressing, Single Cell, Sequencing

Journal: Inflammation

Article Title: IRF7 Modulates Inflammatory Pain Through Upregulating IFNβ in Mice Trigeminal Ganglion

doi: 10.1007/s10753-026-02483-w

Figure Lengend Snippet: Activation of IFNβ signaling contributes to mechanical nociceptive hypersensitivity in mice. ( A ) Schematic illustration of the experimental procedure of Fig. 5 B and C. ( B ) Mechanical hypersensitivity measured by Von Frey filaments in mice intraperitoneally injected with either PBS or IFNβ (10 4 /10 5 U) on day 3 following CFA injection (N = 6). ( C ) The mechanical pain threshold was assessed daily in CFA injected mice using von Frey test, following treatment with either IFNβ (10⁴ U) or PBS (N = 6). ( D - E ) Mechanical hypersensitivity was evaluated by Von Frey test in mice intra-ganglionic injected with an IFNAR1 antagonist (MAR1) or IgG 3 d post-CFA injection. ( F ) Mechanical hypersensitivity of mice receiving direct injection of IFNβ (20 μL) ( G - H ) Representative images of immunofluorescence staining showing c-Fos (red) in the SpVc of mice treated with PBS or IFNβ (N = 5), or mice receiving intra-ganglionically injection of IgG or MAR1(N = 3). (scale bar = 200 μm). ( I - J ) Quantitative analysis of c-Fos (red) expression in the SpVc among groups. Data are expressed as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001; ( B , E ) using one-way ANOVA followed by post hoc test, while ( C , F , I and J ) using unpaired t test

Article Snippet: The IFNAR1 neutralizing antibody MAR1 or mouse IgG (HY- P99137 and HY-P99977 purchased from MedChem Express LLC) was delivered intra-ganglionically on 3 dpi, at a dose of 1 μL(10 μg/1 μL).

Techniques: Activation Assay, Injection, Immunofluorescence, Staining, Expressing

Journal: Inflammation

Article Title: IRF7 Modulates Inflammatory Pain Through Upregulating IFNβ in Mice Trigeminal Ganglion

doi: 10.1007/s10753-026-02483-w

Figure Lengend Snippet: Effects of IRF7-IFNβ signaling on neuronal sensitization and neuroinflammation. ( A ) Detection of cellular calcium changes over time in neurons stimulated with different concentrations of IFNβ. ( B ) Representative fluorescence images showing the expression of Substance P (SP) in neurons following IFNβ stimulation (scale bar = 40 μm). ( C ) mRNA expression levels of CGRP, P2XR, SP, and TRPV1 in mouse TG neurons stimulated with 100 U or 300 U IFNβ, or pretreated with IgG1κ/MAR1 overnight followed by 100 U IFNβ stimulation. ( D - E ) Representative images and quantitative analysis of GFAP and CD86 in the TG of CFA-treated mice after IFNβ treatment (scale bar = 100 μm). ( F ) Western blot analysis of IRF7, NLRP3, IL6 and GAPDH expression in the TG of mice treated with IFNβ or PBS under inflammatory pain conditions. ( G ) Western blot analysis of IRF7, NLRP3, IL6 and GAPDH expression in the TG of CFA- treated mice intra-ganglionically injected with IgG or an IFNAR1 antagonist. ( H ) Western blot analysis of the expression of IRF7, NLRP3, IL6 and GAPDH in the TG of CFA-injected mice receiving sh-Irf7 or sh-Scr stereotaxic microinjection. Data are expressed as mean ± SD, N = 3-7 mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unpaired t test

Article Snippet: The IFNAR1 neutralizing antibody MAR1 or mouse IgG (HY- P99137 and HY-P99977 purchased from MedChem Express LLC) was delivered intra-ganglionically on 3 dpi, at a dose of 1 μL(10 μg/1 μL).

Techniques: Fluorescence, Expressing, Western Blot, Injection, Microinjection

Journal: Journal of Leukocyte Biology

Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

doi: 10.1189/jlb.3A1214-598R

Figure Lengend Snippet: (A) Construct used to generate cd2–ifnar1 transgenic mice. (B) RT-PCR demonstrated lymphoid tissue-specific expression of transgenic ifnar1. With the use of a reverse primer for FLAG, this PCR reaction amplified the ifnar1 transcript only from transgenic mice. Br, Brain; Liv, liver; Lu, lung; Sp, spleen; Th, thymus. (C) RNA was extracted from CD3+-enriched T cells, and ifnar1 mRNA was amplified by real-time RT-PCR. (D) Splenocytes from WT, Ifnar1−/−, and IFNAR1Texcl mice were stained for CD3, CD19, and IFNAR1. (E and F) Splenocytes were stimulated by IFN-α/β (250 U/ml) for 30 min (E) or for the indicated time (F) and then analyzed by Western blot. STAT1 and p-STAT1, 89/91 kDa; ISG15, 15 kDa; actin, 42 kDa. The images were spliced and joined, as indicated by white lines for the sake of the presentation. (G) Thymocytes were incubated with anti-IFNAR1-neutralizing antibody before IFN-α/β treatment and stained with a p-STAT1 antibody. The histogram plots show the p-STAT1-positive cells in the total live-cell gate (IFN treatment, bold, black line; antibody + IFN treatment, black line; no treatment, dotted line).

Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

Techniques: Construct, Transgenic Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Quantitative RT-PCR, Staining, Western Blot, Incubation

Journal: Journal of Leukocyte Biology

Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

doi: 10.1189/jlb.3A1214-598R

Figure Lengend Snippet: (A–C) Splenocytes (A), peritoneal macrophages (B), and LN cells (C) were isolated from WT, Ifnar1−/−, and IFNAR1Texcl mice. Cells were treated with IFN-α/β (250 U/ml) for 15 min and double stained for CD3, CD4, CD8, CD11b, CD19, and p-STAT1. Dot plots show the double-positive cells in the total live-cell gate, and a representative dot plot/group is shown. (D and E) Thymidine uptake after anti (a)-CD3/anti-CD28 stimulation of splenocytes (D) and LN cells (E). Results shown are presented as mean values ± sem. (F) Cytokine production in splenocyte supernatants following stimulation with anti-CD3/anti-CD28. (G) Intracellular staining of splenocytes for IL-2 and IFN-γ after stimulation with anti-CD3/anti-CD28, followed by PMA/ionomycin/brefeldin A incubation. (H and I) CD3+-enriched T cells were stimulated under Th1- and Th17-polarizing conditions and stained for IFN-γ (H) and IL-17A (I). The bars represent the double-positive (CD4+ cytokine+) cells in the total live-cell gate. All data shown are representative of at least 2 independent experiments (n = 5 mice/genotype/experiment).

Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

Techniques: Isolation, Staining, Incubation

Journal: Journal of Leukocyte Biology

Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

doi: 10.1189/jlb.3A1214-598R

Figure Lengend Snippet: (A) Mean clinical scores for WT, Ifnar1−/−, and IFNAR1Texcl mice (n = 9 mice/genotype) after immunization with MOG35–55 peptide. Results shown are representative of 3 independent experiments and are presented as mean values ± sem. (B) Mean body weight of WT (n = 30), Ifnar1−/− (n = 28), and IFNAR1Texcl (n = 32) mice after EAE induction, pooled from 3 independent experiments. Results shown are presented as mean values ± sem.*P < 0.05, **P < 0.01: IFNAR1Texcl versus WT mice; and +P < 0.05, ++P < 0.01: IFNAR1Texcl versus Ifnar1−/− mice.

Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

Techniques:

Journal: Journal of Leukocyte Biology

Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

doi: 10.1189/jlb.3A1214-598R

Figure Lengend Snippet: Clinical severity, day of onset, and peak of MOG 35–55 -induced EAE in IFNAR1 Texcl and control mice

Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

Techniques: Control

Journal: Journal of Leukocyte Biology

Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

doi: 10.1189/jlb.3A1214-598R

Figure Lengend Snippet: Clinical severity of WT and IFNAR1 Texcl mice upon PBS or IFN-β administration

Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

Techniques:

Journal: Journal of Leukocyte Biology

Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

doi: 10.1189/jlb.3A1214-598R

Figure Lengend Snippet: (A–D) Spinal cord sections from WT, Ifnar1−/−, and IFNAR1Texcl mice (n = 3 mice/genotype) were prepared 17 d upon EAE induction and stained with H&E (A), Luxol fast blue (B), and Bielschowsky's silver stain (C). Immunohistochemistry for CD3+ T cells was also performed (D). Arrows indicate immune cell infiltration (A), demyelination (B), axonal damage (C), and CD3+ T cell infiltration in the CNS parenchyma. Scale bars, 1 mm (A–C); 200 μm (D).

Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

Techniques: Staining, Silver Staining, Immunohistochemistry

Journal: Journal of Leukocyte Biology

Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

doi: 10.1189/jlb.3A1214-598R

Figure Lengend Snippet: Splenocytes were isolated from WT, Ifnar1−/−, and IFNAR1Texcl mice, 10 d upon EAE induction. (A) Cells were stimulated with PMA/ionomycin/brefeldin A, and the frequency of IL- 17A-producing CD4+ T cells in the spleen was determined. The dot plots and bars represent IL-17A+ cells gated on total splenocytes. A representative dot plot per genotype is shown. (B) Expression levels of IL-17A by CD4+ T cells were evaluated by measuring MFI from (A) dot plots. (C) Splenocytes from all groups were restimulated ex vivo with MOG35–55 for 72 h, and the levels of secreted IL-17A were measured. (D) Frequency of CCR6+ splenocytes was determined (gated on the CD4+ compartment). All results are shown as means ± sem (n = 5 mice/genotype) and are representative from 2 independent experiments with similar results. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

Techniques: Isolation, Expressing, Ex Vivo

Journal: Journal of Leukocyte Biology

Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

doi: 10.1189/jlb.3A1214-598R

Figure Lengend Snippet: Microarray analysis was performed on RNA from splenic, CD3+-enriched T cells and spinal cords from WT, Ifnar1−/−, and IFNAR1Texcl mice on d 0, 10, and 17 relative to EAE induction. (A and B) Heat map depicting the relative expression of selected genes that are different in expression between IFNAR1Texcl and WT T cells (A) and spinal cord (B) samples and were common in 2 independent experiments. Table 4 shows the function and number of molecules/category, as assessed by DAVID microarray software. The levels of mRNA transcripts for selected genes were measured in T cells (C) and spinal cords (D–F) by quantitative RT-PCR by use of single samples from each group at each time point (d 0: n = 3–5 mice/genotype; d 10: n = 3–4 mice/genotype; d 17: n = 2–3 mice/genotype). The results shown are the means ± sem of samples from 1 representative of 2 independent EAE experiments with similar results.*P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

Techniques: Microarray, Expressing, Software, Quantitative RT-PCR