- 700914 - Invitrogen Antibodies FOXP3 antibody

Submitted references A Biological Circuit Involving Mef2c, Mef2d, and Hdac9 Controls the Immunosuppressive Functions of CD4+Foxp3+ T-Regulatory Cells.

Di Giorgio E, Wang L, Xiong Y, Christensen LM, Akimova T, Han R, Samanta A, Trevisanut M, Brancolini C, Beier UH, Hancock WW
Frontiers in immunology 2021;12:703632

Successful Preclinical Development of Gene Therapy for Recombinase-Activating Gene-1-Deficient SCID.

Garcia-Perez L, van Eggermond M, van Roon L, Vloemans SA, Cordes M, Schambach A, Rothe M, Berghuis D, Lagresle-Peyrou C, Cavazzana M, Zhang F, Thrasher AJ, Salvatori D, Meij P, Villa A, Van Dongen JJM, Zwaginga JJ, van der Burg M, Gaspar HB, Lankester A, Staal FJT, Pike-Overzet K
Molecular therapy. Methods & clinical development 2020 Jun 12;17:666-682

MEF2D sustains activation of effector Foxp3+ Tregs during transplant survival and anticancer immunity.

Di Giorgio E, Wang L, Xiong Y, Akimova T, Christensen LM, Han R, Samanta A, Trevisanut M, Bhatti TR, Beier UH, Hancock WW
The Journal of clinical investigation 2020 Dec 1;130(12):6242-6260

Dataset on the identification of a prognostic radio-immune signature in surgically resected Non Small Cell Lung Cancer.

Mazzaschi G, Milanese G, Pagano P, Madeddu D, Gnetti L, Trentini F, Falco A, Frati C, Lorusso B, Lagrasta C, Minari R, Ampollini L, Silva M, Sverzellati N, Quaini F, Roti G, Tiseo M
Data in brief 2020 Aug;31:105781

Inhibiting the coregulator CoREST impairs Foxp3+ Treg function and promotes antitumor immunity.

Xiong Y, Wang L, Di Giorgio E, Akimova T, Beier UH, Han R, Trevisanut M, Kalin JH, Cole PA, Hancock WW
The Journal of clinical investigation 2020 Apr 1;130(4):1830-1842

Dysfunctional and Proinflammatory Regulatory T-Lymphocytes Are Essential for Adverse Cardiac Remodeling in Ischemic Cardiomyopathy.

Bansal SS, Ismahil MA, Goel M, Zhou G, Rokosh G, Hamid T, Prabhu SD
Circulation 2019 Jan 8;139(2):206-221

Plant Virus-Like Particle In Situ Vaccine for Intracranial Glioma Immunotherapy.

Kerstetter-Fogle A, Shukla S, Wang C, Beiss V, Harris PLR, Sloan AE, Steinmetz NF
Cancers 2019 Apr 10;11(4)

Potentiation of PD-L1 blockade with a potency-matched dual cytokine-antibody fusion protein leads to cancer eradication in BALB/c-derived tumors but not in other mouse strains.

De Luca R, Neri D
Cancer immunology, immunotherapy : CII 2018 Sep;67(9):1381-1391

Cambogin suppresses dextran sulphate sodium-induced colitis by enhancing Treg cell stability and function.

Lu Y, Kim NM, Jiang YW, Zhang H, Zheng D, Zhu FX, Liang R, Li B, Xu HX
British journal of pharmacology 2018 Apr;175(7):1085-1099

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of FOXP3 was performed by loading 30 µg of Jurkat (lane1) and K562 (lane2) cell lysate using NuPAGE® Novex® 10% Bis-Tris gel (Product # NP0301BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature. FOXP3 was detected at ~49 kDa using FOXP3 Recombinant Rabbit Monoclonal Antibody (Product # 700914) at 2-3 µg/mL in 2.5% skim milk at 4°C overnight on a rocking platform. Goat anti-Rabbit IgG - HRP Secondary Antibody (Product # G-21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of FOXP3 in transfected HEK293 cell lysate (30 µg/lane) using a FOXP3 recombinant rabbit monoclonal antibody (Product # 700914) at a dilution of 1 µg/mL. NBT/BCIP was used as the substrate (Product # WB7105).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of FOXP3 showing staining in the nucleus of paraffin-embedded human tonsil tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a FOXP3 monoclonal antibody (Product # 700914) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of FOXP3 showing staining in the nucleus of paraffin-embedded mouse spleen tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a FOXP3 monoclonal antibody (Product # 700914) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of FOXP3 was done on HeLa cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with ABfinity™ FOXP3 Recombinant Rabbit Monoclonal Antibody (700914, red histogram) or with rabbit isotype control (pink histogram) at 3-5 µg/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Extensive Immune Reconstitution of Mice Receiving Gene Therapy of Stem Cells with a Clinical-Grade MND-c.o.RAG1 Vector Rag1-deficient mice were transplanted with 250,000 stem cells: WT cells (three mice), mock KO cells (three mice), and MND-c.o.RAG1-treated cells (VCN of 0.2; eight mice). (A) Representative plots of B cell reconstitution in the blood (B220 + IgM/IgD cells; top panel) and B cell development in the BM (B220 + CD19 + cells; bottom panel) 24 weeks after transplantation. (B) Total number of B cells (B220 + CD11b/CD43 - cells) in the PB (Mann-Whitney test, one-tailed; *p < 0.05, **p < 0.01). (C) Immature (B220 + CD93 + cells; left panel) and mature (B220 + CD93 - cells; right panel) B cell subsets distribution in spleen. Two-way ANOVA test; ***p < 0.001; ****p < 0.0001. (D) Representative plots of T cell reconstitution in the blood (CD3 + TCRab + cells; top panel) and T cell development in the thymus (CD4 versus CD8 cells; bottom panel) 24 weeks after transplantation. (E) Total number of T cells (CD3 + TCRab + cells) in PB at the end of the experiment (24 weeks) (Mann-Whitney test, one-tailed; *p < 0.05, **p < 0.01). (F) Naive, effector memory (EM), and central memory (CM) subset distributions for CD4 (CD3 + TCRab + CD4 + ; left panel) and CD8 (CD3 + TCRab + CD8 + ; right panel). T cell subset distributions in spleen are shown: naive cells (CD44 - CD62L + ), EM cells (CD44 + CD62L - ) and CM cells (CD44 + CD62L + ) 24 weeks after transplantation. (G) Left

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 Cambogin promotes USP7-mediated Foxp3 deubiquitination through Lys-48- and Lys-63-linked deubiquitination. Primary human Treg cells were pretreated with cambogin or USP7 inhibitor, P5091, for 1 h and stimulated with LPS (1 mug*mL -1 ) for 24 h. MG132 was administered for 8 h prior to harvest. Immunoprecipitation was performed as indicated. The relative protein levels were normalized to GAPDH by using ImageJ software. Data were quantified from five independent experiments.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1. Treg expansion in heart failure. A and B , Representative flow cytometry scatter plots and group quantitation for CD3 + CD4 + Foxp3 + Tregs in the heart ( A ) and blood ( B ) from sham and HF mice at the time points indicated. Naive nonsurgical control (NS Ctrl) also shown. C , Representative low and high magnification confocal images of CD4 + (red) and Foxp3 + (green) immunostaining in sham and HF hearts (border, remote, and scar zones 8 weeks after MI) and quantitation of CD4 + Foxp3 + cells. DAPI (blue) was used to label nuclei. D , Representative flow cytometry scatter plots and quantitation for Tregs in the spleen and lymph nodes (LNs) from mice 8 weeks after MI or sham operation. E , Representative flow plots for BrdU in cardiac Tregs ( left ) and quantitation of total BrdU + Tregs in the heart, blood, spleen, and LNs 8 weeks after MI or sham operation ( right ). F , LV (remote zone) and splenic gene expression of IL-2 and IL-6 and IL-2 and IL-6 serum levels in sham and HF mice, respectively (8 weeks after MI). Statistical comparisons: 2-tailed unpaired t test at each time point except for ( A ) and ( B ) ( right ), 2-way ANOVA and Bonferroni posttest performed after ( A ) or without ( B ) logarithmic data transformation. N=4 to 10/group. &ast; P

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