STAT3 [p Tyr705] Antibody [Unconjugated]

Western blot shows lysates of Daudi human Burkitt's lymphoma cell line and HepG2 human hepatocellular carcinoma cell line untreated (-) or treated (+) with 500 U/mL Recombinant Human IFN-aA (Catalog # 11100-1) for 20 minutes or 50 µg/mL Recombinant Human IL-22 (Catalog # 782-IL) for 15 minutes. PVDF membrane was probed with 0.5 µg/mL of Rabbit Anti-Human Phospho-STAT3 (Y705) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4607) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). A specific band was detected for STAT3 at approximately 95 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Daudi human Burkitt's lymphoma cell line was unstimulated (light orange filled histogram) or treated with 500 U/mL rhIFN-alpha for 20 minutes (dark orange filled histogram) was stained with Human Phospho-STAT3 (Y705) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4607) or control antibody (Catalog # AB-105-C, open histogram), followed by Phycoerythrin-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # F0110). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with methanol.

Phospho-STAT3 was detected in immersion fixed IFN-alpha treated Daudi human Burkitt's lymphoma cell line using 10 µg/mL Human Phospho-STAT3 (Y705) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4607) for 3 hours at room temperature. Cells were stained with the NorthernLights™ 557-conjugated Anti-Rabbit IgG Secondary Antibody (red; Catalog # NL004) and counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Simple Western shows lysates of Daudi human Burkitt's lymphoma cell line untreated (-) or treated (+) with 50 µg/mL Recombinant Human IL‑22 (Catalog # 782-IL) for 15 minutes, loaded at 0.2 mg/ml. A specific band was detected for STAT3 at approximately 91 kDa (as indicated) using 10 µg/mL of Rabbit Anti-Human Phospho-STAT3 (Y705) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4607). This experiment was conducted under reducing conditions and using the 12-230kDa separation system.

IL-22BPi1 does not interact with IL-22 or IL-22BPi2. (A) A549 cells were exposed for 30 minutes to dilutions of IL-22-containing culture medium (CM) previously produced by IL22-transfected HEK293 cells. A549 cell lysates (CL) were immunoblotted for pSTAT3 and tubulin as loading control. The relative densitometry of pSTAT3 normalized to that of tubulin is also represented. (B) A549 cells were treated with the optimum IL-22 dilution from A (1/512) for different periods of time, lysed and immunoblotted for pSTAT3. An unspecific protein band (u.p.) was used as loading control. (C) IL-22BP concentration in conditioned medium (CM) of transfected HEK293 cells was measured by ELISA, and the indicated amounts in nanograms (ng) of IL-22BPi1 or IL-22BPi2 were pre-incubated for 1 h at 37°C with the selected IL-22 concentration from (A). A549 were exposed to the pre-incubated combinations for 20 min. An excess of IL-22BPi2 was used as phosphorylation blocking control (lane 3). Cells were lysed and immunoblotted for pSTAT3 and actin as loading control. (D) HeLa cells were co-transfected with the indicated expression plasmids, 24 h later cells were lysed and the CM was subjected to acetone precipitation (AP), and proteins were immunoblotted for FLAG. Intracellular IL-22BPi1 is indicated with dark purple arrows, intracellular and secreted IL-22BPi2 is indicated with green arrows, and co-expressed IL-22 and IL-17 are also indicated. (E) Conditioned media (CM) from 3 independent experiments, in which expression vectors for the three IL-22BP isoforms were individually transfected into HEK293 cells together with either IL-17 or IL-22 expression vectors or an empty vector control (EV), were analyzed 24 h after transfection by ELISA for IL-22BP (mean ± SEM; n = 3; **p < 0.01 by unpaired t-test). (F) IL-22BPi1 does not interact with IL-22BPi2. IL-22BPi1-MF expression plasmid containing Myc and FLAG tags was co-transfected with an inducible pTRE3G-based vector expressing IL-22BPi2 with only a FLAG tag. After 24 h, cells were induced for IL-22BPi2 production by adding Tet-Express activator to the medium for a further 24 h. Cells were lysed and immunoprecipitated with anti-Myc agarose, the flow-through fractions were then further subjected to FLAG immunoprecipitation. CL and eluted fractions were immunoblotted for FLAG. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30619294), licensed under a CC-BY license. Not internally tested by R&D Systems.

IL-22BPi1 does not interact with IL-22 or IL-22BPi2. (A) A549 cells were exposed for 30 minutes to dilutions of IL-22-containing culture medium (CM) previously produced by IL22-transfected HEK293 cells. A549 cell lysates (CL) were immunoblotted for pSTAT3 and tubulin as loading control. The relative densitometry of pSTAT3 normalized to that of tubulin is also represented. (B) A549 cells were treated with the optimum IL-22 dilution from A (1/512) for different periods of time, lysed and immunoblotted for pSTAT3. An unspecific protein band (u.p.) was used as loading control. (C) IL-22BP concentration in conditioned medium (CM) of transfected HEK293 cells was measured by ELISA, and the indicated amounts in nanograms (ng) of IL-22BPi1 or IL-22BPi2 were pre-incubated for 1 h at 37°C with the selected IL-22 concentration from (A). A549 were exposed to the pre-incubated combinations for 20 min. An excess of IL-22BPi2 was used as phosphorylation blocking control (lane 3). Cells were lysed and immunoblotted for pSTAT3 and actin as loading control. (D) HeLa cells were co-transfected with the indicated expression plasmids, 24 h later cells were lysed and the CM was subjected to acetone precipitation (AP), and proteins were immunoblotted for FLAG. Intracellular IL-22BPi1 is indicated with dark purple arrows, intracellular and secreted IL-22BPi2 is indicated with green arrows, and co-expressed IL-22 and IL-17 are also indicated. (E) Conditioned media (CM) from 3 independent experiments, in which expression vectors for the three IL-22BP isoforms were individually transfected into HEK293 cells together with either IL-17 or IL-22 expression vectors or an empty vector control (EV), were analyzed 24 h after transfection by ELISA for IL-22BP (mean ± SEM; n = 3; **p < 0.01 by unpaired t-test). (F) IL-22BPi1 does not interact with IL-22BPi2. IL-22BPi1-MF expression plasmid containing Myc and FLAG tags was co-transfected with an inducible pTRE3G-based vector expressing IL-22BPi2 with only a FLAG tag. After 24 h, cells were induced for IL-22BPi2 production by adding Tet-Express activator to the medium for a further 24 h. Cells were lysed and immunoprecipitated with anti-Myc agarose, the flow-through fractions were then further subjected to FLAG immunoprecipitation. CL and eluted fractions were immunoblotted for FLAG. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30619294), licensed under a CC-BY license. Not internally tested by R&D Systems.

IL-22BPi1 does not interact with IL-22 or IL-22BPi2. (A) A549 cells were exposed for 30 minutes to dilutions of IL-22-containing culture medium (CM) previously produced by IL22-transfected HEK293 cells. A549 cell lysates (CL) were immunoblotted for pSTAT3 and tubulin as loading control. The relative densitometry of pSTAT3 normalized to that of tubulin is also represented. (B) A549 cells were treated with the optimum IL-22 dilution from A (1/512) for different periods of time, lysed and immunoblotted for pSTAT3. An unspecific protein band (u.p.) was used as loading control. (C) IL-22BP concentration in conditioned medium (CM) of transfected HEK293 cells was measured by ELISA, and the indicated amounts in nanograms (ng) of IL-22BPi1 or IL-22BPi2 were pre-incubated for 1 h at 37°C with the selected IL-22 concentration from (A). A549 were exposed to the pre-incubated combinations for 20 min. An excess of IL-22BPi2 was used as phosphorylation blocking control (lane 3). Cells were lysed and immunoblotted for pSTAT3 and actin as loading control. (D) HeLa cells were co-transfected with the indicated expression plasmids, 24 h later cells were lysed and the CM was subjected to acetone precipitation (AP), and proteins were immunoblotted for FLAG. Intracellular IL-22BPi1 is indicated with dark purple arrows, intracellular and secreted IL-22BPi2 is indicated with green arrows, and co-expressed IL-22 and IL-17 are also indicated. (E) Conditioned media (CM) from 3 independent experiments, in which expression vectors for the three IL-22BP isoforms were individually transfected into HEK293 cells together with either IL-17 or IL-22 expression vectors or an empty vector control (EV), were analyzed 24 h after transfection by ELISA for IL-22BP (mean ± SEM; n = 3; **p < 0.01 by unpaired t-test). (F) IL-22BPi1 does not interact with IL-22BPi2. IL-22BPi1-MF expression plasmid containing Myc and FLAG tags was co-transfected with an inducible pTRE3G-based vector expressing IL-22BPi2 with only a FLAG tag. After 24 h, cells were induced for IL-22BPi2 production by adding Tet-Express activator to the medium for a further 24 h. Cells were lysed and immunoprecipitated with anti-Myc agarose, the flow-through fractions were then further subjected to FLAG immunoprecipitation. CL and eluted fractions were immunoblotted for FLAG. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30619294), licensed under a CC-BY license. Not internally tested by R&D Systems.

N-EV and H-EV treatment promote macrophage M2 polarization by delivering miR-21-5p that targets PTEN. a, western blot analysis of PTEN protein expression level in induced macrophages. H/i-miR-EV, monocytes were induced with the presence of EV secreted by miR-21-5p-inhibited, hypoxia pre-challenged MSCs; H-EV + i-miR, monocytes were transfected with miR-21-5p inhibitor-expressing vector before induction with the presence of H-EV. Macrophages induced without MSC-EV were used as negative control (NC). b, c, flow cytometry determining the percentage of CD163+CD206+ cells among total CD68+ cells after induction. N-EV + O/E PTEN or H-EV + O/E PTEN, monocytes were transfected with PTEN overexpressing vector before N-EV or H-EV treatment, respectively. d–f, western blot detecting Akt and STAT3 protein expression as well as their activating phosphorylation (p-Ser473 for Akt and p-tyr705 for STAT3) in macrophages after induction. g–i, ELISA evaluating IL-10, TGF-beta and VEGF-alpha in macrophage culture medium after induction. Macrophages induced with the presence of N-EV were used as negative control in b–i. Tukey’s test was used for statistical analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30736829), licensed under a CC-BY license. Not internally tested by R&D Systems.

• Reactivity: Hu
• Applications: WB, Simple Western, Flow, CyTOF-ready, ICC/IF
• Clonality: Polyclonal
• Host: Rabbit
• Conjugate: Unconjugated
• Concentration: LYOPH

STAT3 [p Tyr705] Antibody [Unconjugated] Summary

• Immunogen: Phosphopeptide containing human STAT3 Y705 site
• Modification: p Tyr705
• Specificity: Detects human STAT3 when phosphorylated at Y705.
• Source: N/A
• Isotype: IgG
• Clonality: Polyclonal
• Host: Rabbit
• Gene: STAT3
• Purity Statement: Antigen Affinity-purified

Applications/Dilutions

• Dilutions:
  • CyTOF-ready
  • Flow Cytometry 0.25 ug/10^6 cells
  • Immunocytochemistry 5-15 ug/mL
  • Simple Western 10 ug/mL
  • Western Blot 0.25-0.5 ug/mL

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.
• Preservative: No Preservative
• Concentration: LYOPH
• Reconstitution Instructions: Reconstitute at 0.2 mg/mL in sterile PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for STAT3 [p Tyr705] Antibody [Unconjugated]

• Acute-phase response factor
• APRFMGC16063
• DNA-binding protein APRF
• FLJ20882
• HIES
• signal transducer and activator of transcription 3 (acute-phase responsefactor)
• signal transducer and activator of transcription 3
• STAT3

Background

Signal Transducer and Activator of Transcription (STAT) proteins are transcription factors activated in response to cytokine, growth factor, or hormone receptor signaling. Janus kinases (JAKs) phosphorylate STAT proteins and induce dimerization. Homo- or heterodimers translocate to the nucleus where they bind to DNA and activate transcription.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for STAT3 Antibody (AF4607)(9)

Publications using AF4607

• Futoon Abedrabbu Al-Rawashde, Abdullah Saleh Al-wajeeh, Mansoureh Nazari Vishkaei, Hanan Kamel M. Saad, Muhammad Farid Johan, Wan Rohani Wan Taib et al. Thymoquinone Inhibits JAK/STAT and PI3K/Akt/ mTOR Signaling Pathways in MV4-11 and K562 Myeloid Leukemia Cells Pharmaceuticals (Basel) 2022-09-08 [PMID: 36145344] (Western Blot, Human)
• Futoon Abedrabbu Al-Rawashde, Abdullah Saleh Al-wajeeh, Mansoureh Nazari Vishkaei, Hanan Kamel M. Saad, Muhammad Farid Johan, Wan Rohani Wan Taib et al. Thymoquinone Inhibits JAK/STAT and PI3K/Akt/ mTOR Signaling Pathways in MV4-11 and K562 Myeloid Leukemia Cells Pharmaceuticals (Basel) 9/8/2022 [PMID: 36145344]
• L Vecchi, STS Mota, MAP Zóia, IC Martins, JB de Souza, TG Santos, AO Beserra, VP de Andrade, LR Goulart, TG Araújo Interleukin-6 Signaling in Triple Negative Breast Cancer Cells Elicits the Annexin A1/Formyl Peptide Receptor 1 Axis and Affects the Tumor Microenvironment Cells, 2022-05-20;11(10):. 2022-05-20 [PMID: 35626741] (Flow Cytometry, Human)
• RP Araldi, TC de Melo, D Levy, DM de Souza, B Maurício, GA Colozza-Ga, SP Bydlowski, H Peng, FJ Rauscher, JM Cerutti LIMD2 Regulates Key Steps of Metastasis Cascade in Papillary Thyroid Cancer Cells via MAPK Crosstalk Cells, 2020-11-23;9(11):. 2020-11-23 [PMID: 33238381] (Flow Cytometry, Human)
• X Chen, R Mao, W Su, X Yang, Q Geng, C Guo, Z Wang, J Wang, LA Kresty, DG Beer, AC Chang, G Chen Circular RNA circHIPK3 modulates autophagy via MIR124-3p-STAT3-PRKAA/AMPK? signaling in STK11 mutant lung cancer Autophagy, 2019-06-28;0(0):1-13. 2019-06-28 [PMID: 31232177] (Western Blot, Human)
• W Ren, J Hou, C Yang, H Wang, S Wu, Y Wu, X Zhao, C Lu Extracellular vesicles secreted by hypoxia pre-challenged mesenchymal stem cells promote non-small cell lung cancer cell growth and mobility as well as macrophage M2 polarization via miR-21-5p delivery J. Exp. Clin. Cancer Res., 2019-02-08;38(1):62. 2019-02-08 [PMID: 30736829] (Western Blot, Human)
• Paloma Gómez-Fernández, Andoni Urtasun, Adrienne W. Paton, James C. Paton, Francisco Borrego, Devin Dersh et al. Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response Frontiers in Immunology 12/14/2018 [PMID: 30619294]
• D. Törőcsik, D. Kovács, E. Camera, M. Lovászi, K. Cseri, G.G. Nagy et al. Leptin promotes a proinflammatory lipid profile and induces inflammatory pathways in human SZ95 sebocytes British Journal of Dermatology 12/1/2014 [PMID: 24975960]
• Kolliputi N, Waxman AB IL-6 cytoprotection in hyperoxic acute lung injury occurs via suppressor of cytokine signaling-1-induced apoptosis signal-regulating kinase-1 degradation. Am. J. Respir. Cell Mol. Biol., 2008-09-05;40(3):314-24. 2008-09-05 [PMID: 18776134] (Western Blot, Human)

Review for STAT3 Antibody (AF4607) (1)

Average Rating: 4

(Based on 1 review)

We have 1 review tested in 1 species: Mouse.

reviewed by: Carme Caelles WB Mouse 04/05/2017

Summary

• Application: Western Blot
• Sample Tested: Whole cell extracts from bone marrow derived macrophages
• Species: Mouse

Bioinformatics

• Gene Symbol: STAT3