14-4774-80

antibody from Invitrogen Antibodies

Targeting: FOXP3 AIID, DIETER, IPEX, JM2, PIDX, SCURFIN, XPID

Provider product page for 14-4774-80

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Antibody data

Antibody Data
Antigen structure
References [26]
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Validations
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Product number: 14-4774-80 - Provider product page
Provider: Invitrogen Antibodies
Product name: FOXP3 Monoclonal Antibody (150D/E4), eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: eBioscience offers a panel of monoclonal antibodies to different epitopes of human/primate and mouse/rat Foxp3 protein, providing useful tools for investigating the complete expression pattern of Foxp3 at the protein level, and discerning the precise subsets of Foxp3+ cells. Other antibodies to Foxp3 available from eBioscience, which have been used significantly in scientific literature, include the anti-human Foxp3 PCH101 (Product # 72-5776) and ebio7979 (Product # 12-7979), and the anti-mouse/rat Foxp3 FJK-16s (Product # 72-5775). The 150D/E4 has been mapped to the splice region in Exon 2 found in human cells. The splicing of this region has not been shown to occur in mouse. Characterization of this antibody to the splice variant has been limited to epitope mapping. It should be noted that other antibodies such as PCH101 and 236A/E7 will recognize both the spliced and full length forms of the Foxp3 protein and stain more intensely than 150D/E4 and FJK-16s in human cells. The 150D/E4 antibody reacts with mouse/rat/human Foxp3 also known as FORKHEAD BOX P3, SCURFIN, and JM2. Cross reactivity of this antibody to other proteins has not been determined. Foxp3, a 49-55 kDa protein, is a member of the forkhead/winged-helix family of transcriptional regulators, and was identified as the gene defective in 'scurfy' (sf) mice. Constitutive high expression of FoxP3 mRNA has been shown in CD4+CD25+ regulatory T cells (Treg cells), and ectopic expression of Foxp3 in CD4+CD25- cells imparts a Treg phenotype in these cells. Applications Reported: This 150D/E4 antibody has been reported for use in immunoblotting (WB), and immunohistochemical staining. Applications Tested: This 150D/E4 antibody has been tested by immunoblotting of recombinant mouse and human Foxp3 protein. The purified format is not recommended for intracellular staining and flow cytometric analysis. Purity: Greater than 90%, as determined by SDS-PAGE. Aggregation: Less than 10%, as determined by HPLC. Filtration: 0.2 µm post-manufacturing filtered.
Reactivity: Human, Mouse, Rat
Host: Mouse
Isotype: IgG
Antibody clone number: 150D/E4
Vial size: 25 µg
Concentration: 0.5 mg/mL
Storage: 4° C

FOXP3 protein structure - 14-4774-80 shown in red.

Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Experimental details: Figure 2. Regulatory T cell (Treg) depletion abrogates atherosclerosis regression. A , Experimental design: Atherosclerosis was established in C57BL6&solJ mice by injection of adeno-associated virus (AAV)-PCSK (proprotein convertase subtilisin&solkexin type)-9 followed by Western diet feeding for 20 wk (Baseline). Mice were then switched to chow diet and atherosclerosis regression was initiated by apoB (apolipoprotein B)-antisense oligonucleotide (ASO)-mediated lipid lowering in conjunction with anti-CD25 or IgG isotype control antibody treatment for 3 wk. B , Plasma total cholesterol, HDL (high-density lipoprotein) and non-HDL cholesterol, and triglyceride levels at baseline and after regression &plus IgG anti-CD25 treatment (n=11 mice&solgroup). C , Representative flow cytometric analyses of CD4 &plus CD25 &plus cells in the circulation of mice treated with IgG or anti-CD25 during atherosclerosis regression. D , Quantification of FoxP3 &plus CD25 &plus cells in peripheral blood (n=5&solgroup), thymus (n=5&solgroup), and spleen (n=9&solgroup) of mice by flow cytometry. Data are mean+-SEM and are representative of 2 independent experiments. P values were determined by a Student t test. E , Representative immunofluorescence staining for FoxP3 in aortic root plaques from mice at baseline and after regression &plus IgG or anti-CD25 treatment (n=6 mice&solgroup). Quantification shown at right. Scale bars=100 &mgrm. F and G , Representative images and quantification of atheroscler

Supportive validation

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Experimental details: Figure 3 Conditional HDAC11 deletion promotes chromatin remodeling at the Foxp3 locus and Treg gene expression. ( A ) ChIP studies of the Foxp3 promoter and 3 enhancer regions in WT vs. conditional HDAC11-/- Treg cells, showing increased acetyl-H3 binding at all 4 sites in HDAC11-/- vs. WT Treg cells. ( B ) Luciferase reporter assays showing the impact of overexpression, and also inhibition, of HDAC11 in 293 T cells that were transiently transfected with murine Foxp3-promoter-Luc vector plus expression vectors for p65, HDAC11, Foxp3, and p300, as specified, or empty vector (EV) along with 50 ng of pRL-TK Renilla luciferase internal control vector; EV was added to ensure equal amounts of DNA per reaction. After 48 h, cells were treated with PMA (6 ng/ml) and ionomycin (1 muM) for 5 to 6 h, and 1 muM HDAC11i where specified, and cell lysates were harvested for luciferase assays. Firefly luminescence data was normalized to Renilla luminescence and analyzed by ANOVA (*p < 0.05, **p < 0.005); data are representative of the results of 3 or more independent experiments. ( C ) Heat-map of selected genes that were upregulated (red) or downregulated (blue) in conditional HDAC11-/- vs. WT Treg cells. ( D ) qPCR evaluation of WT vs. conditional HDAC11-/- Treg cells, showing increased expression of Jun, Junb, Fos and Fosb in HDAC11-/- Tregs; normalized data are shown as mean +- SD, 4 mice/group, and *p < 0.05, **p < 0.01.

Supportive validation

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Experimental details: Fig. 4 mTEC hi -restricted expression of USA preferentially induces T reg differentiation. Littermate control (LMC), CRP, and INS2 Tg mice (CD45.2, Thy1.2) were irradiated and reconstituted with 1% OT-II (CD45.2, Thy1.1) and 99% WT (CD45.1, Thy1.2) bone marrow. The hematopoietic chimeras were analyzed 6-8 weeks post-irradiation. a Proportion of mature TCRbeta hi CD4 + SP OT-II thymocytes among total live cells and absolute number of live CD4 + SP OT-II thymocytes. b Proportion of OT-II cells within the CD4 + T cell population in the peripheral lymph nodes. c Representative flow plots of CD25 and FoxP3 expression on OT-II T cells. d Proportion and absolute cell number of CD25 + Foxp3 + OT-II T cells in the thymus. e Proportion of CD25 + Foxp3 + OT-II T cells in the lymph nodes. (mean +- SEM, n = 17 LMC, 8 CRP, 10 INS2, from three independent experiments). All p -values indicated are calculated by Kruskal-Wallis test followed by Dunn's post-hoc comparisons to LMC, two-sided. ** p < 0.01 relative to LMC. Source data are provided as a Source Data file.

Supportive validation

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Experimental details: Fig. 5 TRA expression by distinct mTEC compartments impacts the mode of tolerance induction in the polyclonal T cell repertoire in naive mice. a Representative flow plots of H-2K b -OVA tetramer on pooled peripheral lymph nodes and spleen cells from naive mice after enrichment, gated on live TCRalphabeta + CD8 + T cells. Relative number of H-2K b -OVA-specific TCRalphabeta + CD8 + T cells in the peripheral secondary lymphoid organs (mean +- SEM, n = 12 LMC, 7 CRP, 7 INS2) ( b ) and intraepithelial lymphocytes (IEL) (mean +- SEM, n = 17 LMC, 11 CRP, 8 INS2) ( c ) from CRP and INS2 Tg mice as compared to their respective littermate controls (LMC). d Representative flow plot of CD8alpha and CD8beta and e relative proportion of TCRalphabeta + CD8alphaalpha within the H-2K b -OVA tetramer double positive TCRalphabeta IEL of CRP and INS2 Tg mice as compared to their respective LMC (mean +- SEM, n = 17 LMC, 11 CRP, 8 INS2). f Representative flow plots of I-A b -gp66 tetramer on pooled peripheral lymph nodes and spleen cells from naive mice, gated on live TCRalphabeta + CD4 + T cells. g Relative number of I-A b -gp66 tetramer DP CD4 + T cells in the peripheral lymph nodes and the spleen of naive mice. h Representative flow plots of CD25 and FoxP3, gated on live I-A b -gp66 tetramer + CD4 + T cells. i Relative proportion of CD25 + FoxP3 + T reg of gp66-specific CD4 + T cells and relative number of gp66-specific CD4 + CD25 + FoxP3 + T reg from CRP and INS2 peripheral lymph nodes and sp

Supportive validation

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Experimental details: Fig. 6 TRA expression by distinct mTEC compartments impacts the mode of tolerance induction in the polyclonal T cell repertoire in LCMV infected mice. Mice were infected with LCMV-OVA and the antigen-specific response was measured in the spleen 8 days post-infection (NI, not infected mice) a, c Representative CD44 and H-2K b -OVA or H-2D b -gp33 tetramer staining on CD8 + T cells from littermate controls (LMC), CRP, and INS2 Tg mice. Relative response and absolute number of OVA- ( b ) and gp33- ( d ) specific CD8 + T cell response in USA expressing mice as compared to LMC. e Representative CD44 and I-A b -gp66 tetramer staining on CD4 + T cells from LMC, CRP, and INS2 Tg mice. f Relative and absolute gp66-specific response in USA expressing mice. g Representative flow plot of CD25 and FoxP3, gated on live I-A b -gp66-specific CD4 + T cells. h Relative proportion of CD25 + FoxP3 + T reg among gp66-specific CD4 + T cells and absolute number of gp66-specific CD4 + CD25 + FoxP3 + T reg (mean +- SEM, b left panel n = 22 LMC, 7 CRP, 5 CRP lo , 9 INS2, 3 INS2 lo ; b right panel n = 3 NI, 8 CRP LMC, 10 CRP Tg, 9 CRP lo LMC, 9 CRP lo Tg, 10 INS2 LMC, 9 INS2 Tg, 3 INS2 lo LMC, 3 INS2 lo Tg; d and f left panel n = 32 LMC, 12 CRP, 12 CRP lo , 14 INS2, 8 INS2 lo , d right panel n = 6 NI, 11 CRP LMC, 12 CRP Tg, 11 CRP lo LMC, 12 CRP lo Tg, 13 INS2 LMC, 11 INS2 Tg, 6 INS2 lo LMC, 6 INS2 lo Tg, f right panel n = 6 NI, 11 CRP LMC, 13 CRP Tg, 12 CRP lo LMC, 13 CRP lo Tg, 14 INS2 LMC, 12 INS2 T