mouse monoclonal anti phospho rela nfkb p65 s529  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: BTLA agonist attenuates Th17-driven inflammation in a mouse model of steroid-resistant asthma

doi: 10.3389/fimmu.2025.1552394

Figure Lengend Snippet: HDM-driven airway inflammation in BXD75 mice are enriched for genes related to Th17 mediated inflammation. (A) Volcano plot of differentially expressed genes (DEGs) in C57BL/6 vs BXD75 mice challenged with HDM only. (B) Gene set enrichment analysis by Ingenuity Pathway Analysis (IPA) depicting enriched pathways in HDM-challenged BXD75 mice. (C) Heatmap representation of co-receptors in CD4 + T cells isolated from BXD75 mice vs C57BL/6 mice challenged with HDM. n = 3 mice per genotype. Mean fluorescence intensity (MFI) and representative flow cytometry histograms of (D) HVEM, (E) LIGHT, and (F) BTLA in CD4 + T cells from C57BL/6 and BXD75 mice challenged with HDM. (G) Schematic of downstream HVEM-LIGHT stimulatory signaling in T cells. MFI of (H) TRAF2, (I) p65, (J) p52, and (K) NIK in CD4 + T cells from HDM or PBS challenged C57BL/6 and BXD75 mice. Data in (D–F) are from one experiment that is representative of three independent experiments. Data in (H–K) are pooled from two experiments. Each experiment was performed at least three times. For all quantifications, data are presented as mean ± SEM and analyzed with a two-way ANOVA with Tukey’s multiple comparison test. n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ***, p < 0.0001.

Article Snippet: Staining for the NFkB pathway was done with the following antibodies: PE-anti-mouse TRAF2 (Clone H-10, Santa Cruz Biotechnology), PE anti-mouse NIK (Clone A-12, Santa Cruz Biotechnology), Alexa ® Fluor 647 anti-mouse NFκB p52/p100/NFKB2 Antibody (Clone C-5, Santa Cruz Biotechnology), PE anti-human/mouse p65/RelA (Clone 532301, R&D Systems).

Techniques: Isolation, Fluorescence, Flow Cytometry, Comparison

Journal: Frontiers in Immunology

Article Title: BTLA agonist attenuates Th17-driven inflammation in a mouse model of steroid-resistant asthma

doi: 10.3389/fimmu.2025.1552394

Figure Lengend Snippet: BTLA agonist treatment attenuates Th17 activation in vitro . (A) Naïve CD3 + CD4 + CD44 low T cells were isolated from C57BL/6 mice and cultured with bound anti-CD3, soluble CD28, rmIL-6, rmTGF- β1, rmIL-23, anti-IL-12, anti-IL-4 and anti-IFNγ for 5 days. On day 3, cells were treated with 20 μg/mL of BTLA agonist (Clone 6A6) or Isotype. (B) The number of CD4 + IL - 17A + T cells derived from C57BL/6 mice. (C) Levels of IL-17A in the cell culture supernatant. (D) Representative flow cytometry histogram and (E) quantification of SHP-1 expression MFI on Day 5. (F) Representative flow cytometry histogram and (G) quantification of p65 expression MFI on Day 5. (H) Representative flow cytometry histogram and (I) quantification of NIK expression MFI on Day 5. (J) Representative flow cytometry histogram and (K) quantification of p52 expression MFI on Day 5. Histograms are from one experiment that is representative of three independent experiments. Data in (B, C, E, I) are pooled from two experiments. Data in (G, K) are from one representative experiment. Each experiment was performed at least three times. For all quantifications, data are presented as mean ± SEM and analyzed with a two-tailed Student’s t test. n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ***, p < 0.0001.

Article Snippet: Staining for the NFkB pathway was done with the following antibodies: PE-anti-mouse TRAF2 (Clone H-10, Santa Cruz Biotechnology), PE anti-mouse NIK (Clone A-12, Santa Cruz Biotechnology), Alexa ® Fluor 647 anti-mouse NFκB p52/p100/NFKB2 Antibody (Clone C-5, Santa Cruz Biotechnology), PE anti-human/mouse p65/RelA (Clone 532301, R&D Systems).

Techniques: Activation Assay, In Vitro, Isolation, Cell Culture, Derivative Assay, Flow Cytometry, Expressing, Two Tailed Test

Journal: Journal of Clinical Investigation

Article Title: FOXO1 pathway activation by VISTA immune checkpoint restrains pulmonary ILC2 functions

doi: 10.1172/jci184932

Figure Lengend Snippet: Figure 5. FOXO1 pathway activation by VISTA regulates ILC2 function. (A) Overview of the VISTA downstream signaling. (B-E) Freshly isolated WT and Vsir-/- aILC2s were stained intranuclearly for pAKT, FOXO1, p65 and p52. Representative histogram of protein expression of pAKT (B), FOXO1 (C), p65 (D), p52(E), and corresponding quantitations as MFI. (F-I) WT aILC2 were treated with FOXO1 inhibitor or vehicle. (G-H) Representative plots of GATA-3 (G) and Ki67 (H) expression levels and corresponding quantification (as MFI). (I) Cytokine levels in cell supernatant (per 103 ILC2s). (J-M) WT aILC2 were treated with FOXO1 activator or vehicle. (K-L) Representative plots of GATA-3 (K) and Ki67 (L) expression levels and corresponding quantification (as MFI). (M) Cytokine levels in cell supernatant (per 103 ILC2s). Data are presented as mean+SEM and are representative of at least two independent experiments. Statistical significance was assessed using a two-tailed Student's t-test; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Anti-p65 (IC5078P, PE) was obtained from R&D Systems, and anti-p52 (C-5, PE) was purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Isolation, Staining, Expressing, Two Tailed Test

Journal: Cellular and Molecular Immunology

Article Title: SIRPα engagement regulates ILC2 effector function and alleviates airway hyperreactivity via modulating energy metabolism

doi: 10.1038/s41423-024-01208-z

Figure Lengend Snippet: SIRPα modulates ILC2 mitochondrial respiration via NF-κB pathways. A – P Cohorts of WT and SIRPα KO mice were intranasally challenged with rmIL-33 over 3 consecutive days. On day 4, lung ILC2s were isolated and cultured with rmIL-2 and rmIL-7 for 24 h. B Total RNA was extracted to perform a bulk transcriptomic analysis. Volcano plots represent differentially expressed genes. C Gene set enrichment analysis by Ingenuity Pathway Analysis (IPA) depicting critical pathways regulated in SIRPα-deficient ILC2s. D Dot plot representation of selected critical genes involved in ILC2 related genes, MAPK, and JAK/STAT pathways. Dot size is indicative of the total gene expression level. E Overview of downstream SIRPα signaling elements. F – I Cohorts of WT and SIRPα KO mice were challenged intranasally for 3 days with rmIL-33. On day 4, lung ILC2s were isolated and cultured with rmIL-2, rmIL-7 for 24 h. Representative histogram of protein expression of pSTAT3 ( G ), p38 ( H ), KLF2 ( I ), and p65 ( J ). Corresponding quantitation is presented as MFI; n = 4. Corresponding quantitation is presented for each protein as MFI; n = 4. K Dot plot representation of selected critical genes involved in OXPHOS and Mitochondrial respiratory pathways. Dot size is indicative of the total gene expression level. L – O Mitochondrial respiratory profile showing oxygen consumption rates (OCR) in response to sequential injections of Oligomycin (ATP synthase inhibitor), BAM15 (mitochondrial uncoupler), and Rotenone + antimycin A (complex I and II inhibitors). Key parameters of mitochondrial function, including basal respiration ( M ), spare respiratory capacity ( N ), and ATP production rate ( O ) are presented; n = 3. P Mitochondrial sizes were assessed using Mito Tracker green and are shown in plot graphs. Data are presented as mean +/− SEM and are representative of at least 2 experiments. Two-tailed student’s t-test was employed for statistical analysis; *< 0.05, **< 0.01, ***< 0.001, and ns= non-significant. Schematic images are sourced by an open-access license from Servier Medical Art

Article Snippet: Phosphorylation assessment involved FITC anti-human/mouse Phospho STAT3 (RUO, Biosciences), PE anti-human/mouse Rela (p65) (532301, R&D systems), APC anti-human/mouse Phospho p38 (4NIT4KK, Invitrogen), and Alexa FluorTM 647 anti-human/mouse KLF2 (bs-2772R, Bioss).

Techniques: Isolation, Cell Culture, Gene Expression, Expressing, Quantitation Assay, Two Tailed Test

Journal: Cellular and Molecular Immunology

Article Title: SIRPα engagement regulates ILC2 effector function and alleviates airway hyperreactivity via modulating energy metabolism

doi: 10.1038/s41423-024-01208-z

Figure Lengend Snippet: CD47 administration controls ILC2 function via activating SIRPα signaling. A )WT (CD45.1) and CD47 KO (CD45.2) mice were intranasally exposed to 0.5 μg rmIL-33 for three days, and lung ILC2s were isolated on day four. Two groups were formed: one consisting ILC2s from only CD47 KO mice (monoculture group), and another consisting of ILC2s from both CD47 KO (CD45.2) and WT (CD45.1) mice, which were co-cultured in a 1:1 ratio (co-culture group) and incubated for 24 h. The function of CD47 KO-derived ILC2 in each group was then evaluated by FACS. B , C Expression levels of GATA-3 ( B ) and Ki67 ( C ) in activated ILC2s are presented. Corresponding quantitation is presented as MFI; n = 4. D Frequency (%) of IL-5 + and IL-13 + ILC2s in both monoculture and co-culture groups; n = 4. E – I WT mice received intranasal doses of rmIL-33 over 3 consecutive days. Activated ILC2s were sorted and cultured with rmIL-2 (10 ng/ml) and rmIL-7 (10 ng/ml) in the presence of either vehicle or CD47-Fc (20 μg/mL) for 24 h. F , G GATA-3 ( F ) and Ki67 ( G ) expression levels in activated ILC2s are shown. Corresponding quantitation is presented as MFI; n = 4. H , I Levels of IL-5 ( H ) and IL-13 ( I ) production in the culture supernatant were measured by LEGENDPLEX and are shown in bar graphs; n = 4. J WT mice were treated with rmIL-33 by intranasal injection for three days in a row. Activated ILC2 cells were sorted and cultured with rmIL-2 (10 ng/ml) and rmIL-7 (10 ng/ml). The following day, vehicle or CD47-Fc (20 μg/mL) was added to the culture wells and analyzed by FACS after one hour. K Overview of downstream SIRPα signaling elements. L – O Representative histogram of protein expression of pSTAT3 ( L ), p38 ( M ), KLF2 (N ), and p65 ( O ). Corresponding quantitation is presented for each protein as MFI; n = 4. Data are presented as means ± SEM and are representative of at least 2 independent experiments. Two-tailed student’s t-test or one-way ANOVA followed by Tukey post-hoc tests were employed for statistical analysis; *< 0.05, **< 0.01, ***< 0.001, and ****< 0.0001. Schematic images are sourced by an open access license from Servier Medical Art

Article Snippet: Phosphorylation assessment involved FITC anti-human/mouse Phospho STAT3 (RUO, Biosciences), PE anti-human/mouse Rela (p65) (532301, R&D systems), APC anti-human/mouse Phospho p38 (4NIT4KK, Invitrogen), and Alexa FluorTM 647 anti-human/mouse KLF2 (bs-2772R, Bioss).

Techniques: Isolation, Cell Culture, Co-Culture Assay, Incubation, Derivative Assay, Expressing, Quantitation Assay, Injection, Two Tailed Test