25-9033-42

antibody from Invitrogen Antibodies

Targeting: STAT3 APRF

Flow cytometry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

25-9033-42

Provider

Invitrogen Antibodies

Product name

Phospho-STAT3 (Tyr705) Monoclonal Antibody (LUVNKLA), PE-Cyanine7, eBioscience™

Antibody type

Monoclonal

Antigen

Other

Description

Description: This LUVNKLA monoclonal antibody recognizes human and mouse signal transducer and activator of transcription 3 (STAT3) when phosphorylated on tyrosine 705 (Y705). The STAT family represents seven transcription factors (STATs 1, 2, 3, 4, 5A, 5B, and 6) that are involved in many cellular processes including apoptosis, cell differentiation, and proliferation in a cell type- and cytokine-specific manner. STAT proteins are activated by ligand binding to cytokine receptors that associate with Janus kinase (JAK) family members.

Antibody clone number

LUVNKLA

Concentration

5 µL/Test

References

Submitted references

Combinatorial immunotherapy of N-803 (IL-15 superagonist) and dinutuximab with ex vivo expanded natural killer cells significantly enhances in vitro cytotoxicity against GD2(+) pediatric solid tumors and in vivo survival of xenografted immunodeficient NSG mice.

Chu Y, Nayyar G, Jiang S, Rosenblum JM, Soon-Shiong P, Safrit JT, Lee DA, Cairo MS
Journal for immunotherapy of cancer 2021 Jul;9(7)

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Mapping of Signaling Pathways Linked to sIgAD Reveals Impaired IL-21 Driven STAT3 B-Cell Activation.

Lemarquis AL, Theodors F, Einarsdottir HK, Ludviksson BR
Frontiers in immunology 2019;10:403

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CD1d highly expressed on DCs reduces lung tumor burden by enhancing antitumor immunity.

Li Y, Zhao C, Liu J, Lu Z, Lu M, Gu J, Liu R
Oncology reports 2019 May;41(5):2679-2688

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ATF3 Sustains IL-22-Induced STAT3 Phosphorylation to Maintain Mucosal Immunity Through Inhibiting Phosphatases.

Glal D, Sudhakar JN, Lu HH, Liu MC, Chiang HY, Liu YC, Cheng CF, Shui JW
Frontiers in immunology 2018;9:2522

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Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation.

Ferrao RD, Wallweber HJ, Lupardus PJ
eLife 2018 Jul 25;7

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Intracellular staining of untreated (left) or 15-minute IL-6 treated (right) normal human peripheral blood cells with Anti-Human CD4 eFluor® 450 (Product # 48-0049-42) and Anti-Human/Mouse phospho-STAT3 (Y705) PE-Cyanine7. Cells in the lymphocyte gate were used for analysis. Cells were stained using the Intracellular Fixation/Methanol protocol.

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 5. EPOR and LEPR switch residues are required for Epo and Leptin-induced STAT phosphorylation. ( a,b ) Stable Ba/F3 cell lines expressing wild-type, full length mouse EPOR or LEPR were generated and analyzed for STAT phosphorylation by flow cytometry after stimulation with EPO or leptin. ( a ) Representative plots comparing phospho-STAT5 staining of parental Ba/F3 cells or Ba/F3 cells expressing EPOR. Cells were stimulated with 1 nM mouse EPO for 15 min before fixation, staining, and analysis. ( b ) Representative plots comparing phospho-STAT3 staining of parental Ba/F3 cells or Ba/F3 cells expressing LEPR. Cells were stimulated with 100 pM mouse Leptin for 4 hr prior to fixation, staining, and analysis. ( c ) Analysis of STAT5 phosphorylation by flow cytometry for EPOR wild-type, switch region, and box1 mutants, stimulated with 1 nM EPO as in ( a ). Mean levels of STAT5 phosphorylation were assessed in three separate experiments, with wild-type signal representing 100% in all three experiments. ( d ) Analysis of STAT3 phosphorylation by flow cytometry for LEPR wild-type, switch region, and box1 mutants, stimulated with 100 pM Leptin, as in ( b ). Mean levels of STAT3 phosphorylation was assessed in three separate experiments, with wild-type signal representing 100% in all three experiments. Error bars represent standard error of the mean (SEM). Figure 5--figure supplement 1. Analysis of Ba/F3 stable cell lines expressing EPOR or LEPR mutants. ( a ) Alignment of human

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 5--figure supplement 1. Analysis of Ba/F3 stable cell lines expressing EPOR or LEPR mutants. ( a ) Alignment of human and mouse sequences for EPOR and LEPR. Note the numbering differences for mouse versus human (minus one residue for EPOR and minus two residues for LEPR). Residues in the switch region and box1, which are mutated in our experiments, are completely conserved between human and mouse. ( b-c ) Receptor surface staining for stable cell lines expressing ( b ) EPOR variants and ( c ) LEPR variants. Plots represent cumulative events (Y axis) at or below a given staining level (X axis). ( d ) Representative raw flow cytometry histograms showing the phospho-STAT5 (EPOR) or phospho-STAT3 (LEPR) staining in unstimulated cells (black trace) or cells stimulated with cytokine (red trace) as described in Materials and methods.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 1 N-803 increased the viability and proliferation of exPBNK with enhanced p-Stat3, p-Stat5, pAKT, p-p38MAPK and NK activating receptors. PBMNCs were stimulated with irradiated genetically modified K562-mbIL21-41BBL cells for 2-3 weeks. (A) Purified exPBNK cells were cultured in complete medium with 0.35 ng/mL (low) or 3.5 ng/mL (high) N-803 or molar equivalent dose of IgG for 3 days. NK viability and proliferation were monitored by MTS assays. The amount of 490 nm absorbance is directly proportional to the number of living exPBNK cells in the culture. The exPBNK cells with N-803 at 0.35 ng/mL or 3.5 ng/mL have significantly higher viability as compared with IgG or medium controls (p

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 7 ATF3 promotes IL-22-induced STAT3 phosphorylation by suppressing phosphatases. (A) Freshly isolated ileum crypts, or (B) ileum organoids at day 6 of culture, were stimulated with IL-22, followed by fixation and intracellular staining of phospho-STAT3, and analyzed by flow cytometry. Western blot analysis of (C) IL-22-stimulated CMT93 cells, or (D) IL-22-stimuated colon fragments isolated from the indicated mice, for the expression of the indicated proteins. (E) Quantitative real-time PCR analysis of IL-22R1 and IL-10R2 mRNA levels in freshly isolated ileum crypts from mice. (F) Flow cytometry analysis of IL-22R1 in freshly isolated ileum crypt cells gated on the CD45 - EpCAM + population. (G,H) Western blot analysis of unstimulated or IL-22-stimulated CMT93 cells for the indicated proteins. ATF3 -/- CMT93 cells with SHP2 knockdown (ATF3 -/- SHP2 KD ) were indicated. Images were representative of four independent experiments (G-H) . Results were from two independent experiments (A-F) . ""n"" refers to the number of mice analyzed (A,B,E,F) . Statistical analysis was done by multiple comparison in Two-way ANOVA test using Prism software. * P < 0.05, ** P < 0.005, *** P < 0.0005.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

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Experimental details

Figure 8 ATF3 regulates IL-6-pSTAT3 signaling in intestinal Th17 cells. Flow cytometry analysis of phospho-STAT3 in (A) IL-6 or IL-22 stimulated freshly isolated ileum crypts or IL-6-stimulated peripheral blood mononuclear cell (PBMC) from wild-type mice, or in (B) IL-6-stimulated PBMC from wild-type and ATF3-deficient mice. (C) Flow cytometry analysis of intracellular IL-17A and IL-22 expression in naive lamina propria T cells from the indicated mice. Cells were treated with PMA, ionomycin and IL-23 in the presence of BFA for 4 h before analysis and gated on live CD45 + EpCAM - Lin - CD3 + population as shown. (D) Quantitative real-time PCR analysis of IL-17A and IL-22 mRNA levels in freshly isolated lamina propria (LPL) cells, mesenteric lymph nodes (mLN), or splenocytes. (E) Model of ATF3-mediated mucosal immunity via cross-regulation between IL-22-pSTAT3 signaling in epithelium (associated with AMP production and epithelial fucosylation) and IL-6-pSTAT3 signaling in Th17 cells (associated with signature IL-17A and IL-22 production). ""n"" refers to the number of mice analyzed. Statistical analysis was done by multiple comparison in Two-way ANOVA test using Prism software. * P < 0.05, ** P < 0.005, *** P < 0.0005.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

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Experimental details

Figure 5. (A) The correlation analysis between CD1d + DC cell score and Co antigen-presenting activity score. The costimulatory molecule scores were calculated by combining the expression levels of CD40, CD80 and CD86; the MHC-I score was calculated by combining the expression levels of HLA-A/B/C; and the MHC-II score was calculated by combining the expression levels of HLA-DMA/DOA/DQA1/DRA. The co antigen-presenting activity score was calculated by combining the expression levels of CD40, CD80, CD86 and MHC-I/II. Pearsons correlation coefficient was performed. (B) Protein levels of pp65, pERK1/2 and pJAK2 in CD1d + DC cells and CD1d - DC cells were detected via western blot analysis. FACS analysis of pSTAT6 and pSTAT3 in CD1d + DC cells and CD1d - DC cells. CD1d + DC cells were treated with inhibitors of pSTAT3 (10 nM) and pSTAT6 (10 nM), and then expression levels of CD1d were detected by FACS. DC, dendritic cells; MFI, median fluorescence intensity; FACS, fluorescence-activated cell sorter.