Key Product Details

Validated by

Biological Validation

Species Reactivity

Validated:

Human

Cited:

Human, Mouse

Applications

Validated:

Western Blot, Flow Cytometry, Immunocytochemistry, Simple Western, CyTOF-ready

Cited:

Western Blot, Flow Cytometry

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG
View all STAT3 Primary Antibodies »

Product Specifications

Immunogen

Phosphopeptide containing human STAT3 Y705 site

Specificity

Detects human STAT3 when phosphorylated at Y705.

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Scientific Data Images for Human Phospho-STAT3 (Y705) Antibody

Detection of Human Phospho-STAT3 (Y705) antibody by Western Blot.

Detection of Human Phospho-STAT3 (Y705) by Western Blot.

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.

Detection of Phospho-STAT3 (Y705) antibody in IFN-alpha-treated Human Daudi Cell Line antibody by Flow Cytometry.

Detection of Phospho-STAT3 (Y705) in IFN-alpha-treated Human Daudi Cell Line by Flow Cytometry.

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.

STAT3 antibody in Daudi Human Burkitt's Lymphoma Cells by Immunocytochemistry (ICC).

STAT3 in Daudi Human Burkitt's Lymphoma Cells.

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.

Detection of Human STAT3 by Simple WesternTM.

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.

Detection of Human STAT3 by Western Blot

Detection of Human STAT3 by Western Blot

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 (https://pubmed.ncbi.nlm.nih.gov/30619294), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human STAT3 by Western Blot

Detection of Human STAT3 by Western Blot

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 (https://pubmed.ncbi.nlm.nih.gov/30619294), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human STAT3 by Western Blot

Detection of Human STAT3 by Western Blot

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 (https://pubmed.ncbi.nlm.nih.gov/30619294), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human Human Phospho-STAT3 (Y705) Antibody by Western Blot

Detection of Human Human Phospho-STAT3 (Y705) Antibody by Western Blot

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 (https://pubmed.ncbi.nlm.nih.gov/30736829), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Phospho-STAT3 (Y705) by Flow Cytometry

Detection of Phospho-STAT3 (Y705) by Flow Cytometry

Interleukin 6 (IL-6) and STAT3 expression in BC cells. (A) IL-6 levels were measured by ELISA assay in supernatants of MCF-10, MCF-7, MDA-MB-231, BT-549, MDA-MB-157 and MDA-MB-453 cells. IL-6 mRNA relative levels of MCF-10, MCF-7, MDA-MB-231, BT-549, MDA-MB-157 and MDA-MB-453 cells were also recorded. (B) Expression of activated STAT3 (STAT3pY705) in MCF-10, MCF-7, MDA-MB-231, BT-549, MDA-MB-157 and MDA-MB-453 cells was analyzed by flow cytometry (gray peak). As the negative control, the secondary antibody alone was used (white peak). (C) STAT3 mRNA relative levels of MCF-10, MCF-7, MDA-MB-231, BT-549, MDA-MB-157 and MDA-MB-453 cells. (D) STAT3 phosphorylation levels (STAT3pY705-dark gray peak with tracing line) were analyzed in MDA-MB-231 and MDA-MB-157 cells treated with Tocilizumab (TCZ), recombinant IL-6 (rIL-6) and STAT3 inhibitor, STATTIC (STC). As the negative control, the secondary antibody alone was used (white peak). * p < 0.05; ** p < 0.001. Three independent experiments were performed. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35626741), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Phospho-STAT3 (Y705) by Flow Cytometry

Detection of Phospho-STAT3 (Y705) by Flow Cytometry

Interleukin 6 (IL-6) and STAT3 expression in BC cells. (A) IL-6 levels were measured by ELISA assay in supernatants of MCF-10, MCF-7, MDA-MB-231, BT-549, MDA-MB-157 and MDA-MB-453 cells. IL-6 mRNA relative levels of MCF-10, MCF-7, MDA-MB-231, BT-549, MDA-MB-157 and MDA-MB-453 cells were also recorded. (B) Expression of activated STAT3 (STAT3pY705) in MCF-10, MCF-7, MDA-MB-231, BT-549, MDA-MB-157 and MDA-MB-453 cells was analyzed by flow cytometry (gray peak). As the negative control, the secondary antibody alone was used (white peak). (C) STAT3 mRNA relative levels of MCF-10, MCF-7, MDA-MB-231, BT-549, MDA-MB-157 and MDA-MB-453 cells. (D) STAT3 phosphorylation levels (STAT3pY705-dark gray peak with tracing line) were analyzed in MDA-MB-231 and MDA-MB-157 cells treated with Tocilizumab (TCZ), recombinant IL-6 (rIL-6) and STAT3 inhibitor, STATTIC (STC). As the negative control, the secondary antibody alone was used (white peak). * p < 0.05; ** p < 0.001. Three independent experiments were performed. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35626741), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Phospho-STAT3 (Y705) by Western Blot

Detection of Phospho-STAT3 (Y705) by Western Blot

Aspirin modulated the JAK/p-STAT3 signaling pathway in atypical hyperplastic intestinal mucosal cells of UC mice (n = 4 for each group). (A) Western blotting images for JAK/p-STAT3 signaling pathway-associated molecules expression. (B) Statistical analysis and comparison for p-STAT3 expression. (C) Statistical analysis and comparison for STAT3 expression. (D) Statistical analysis and comparison for cyclin D1 expression. (E) Statistical analysis and comparison for SOCS3 expression. *P <.05 versus control group. #P <.05 versus UC model group. JAK, Janus kinase; UC, ulcerative colitis; p-STAT3, phosphorylated-STAT3; STAT3, signal transducer and activator of transcription 3; SOCS3, suppressor of cytokine signaling 3. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35946886), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Phospho-STAT3 (Y705) by Western Blot

Detection of Phospho-STAT3 (Y705) by Western Blot

Aspirin modulated the JAK/p-STAT3 signaling pathway in atypical hyperplastic intestinal mucosal cells of UC mice (n = 4 for each group). (A) Western blotting images for JAK/p-STAT3 signaling pathway-associated molecules expression. (B) Statistical analysis and comparison for p-STAT3 expression. (C) Statistical analysis and comparison for STAT3 expression. (D) Statistical analysis and comparison for cyclin D1 expression. (E) Statistical analysis and comparison for SOCS3 expression. *P <.05 versus control group. #P <.05 versus UC model group. JAK, Janus kinase; UC, ulcerative colitis; p-STAT3, phosphorylated-STAT3; STAT3, signal transducer and activator of transcription 3; SOCS3, suppressor of cytokine signaling 3. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35946886), licensed under a CC-BY license. Not internally tested by R&D Systems.

Applications for Human Phospho-STAT3 (Y705) Antibody

Application
Recommended Usage

CyTOF-ready

Ready to be labeled using established conjugation methods. No BSA or other carrier proteins that could interfere with conjugation.

Flow Cytometry

0.25 µg/106 cells
Sample: Daudi human Burkitt's lymphoma cell line treated with IFN-alpha, fixed with paraformaldehyde and permeabilized with methanol

Immunocytochemistry

5-15 µg/mL
Sample: Immersion fixed IFN-alpha treated Daudi human Burkitt's lymphoma cell line

Simple Western

10 µg/mL
Sample: Daudi human Burkitt's lymphoma cell line untreated (-) or treated (+) with 50 µg/mL Recombinant Human IL-22 (Catalog # 782-IL) for 15 minutes

Western Blot

0.25-0.5 µg/mL
Sample: Daudi human Burkitt's lymphoma cell line treated with Recombinant Human IFN‑ alpha A (Catalog # 11100-1) and HepG2 human hepatocellular carcinoma cell line treated with Recombinant Human IL‑22 (Catalog # 782-IL)

Reviewed Applications

Read 3 reviews rated 4.7 using AF4607 in the following applications:

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Flow Cytometry

Formulation, Preparation, and Storage

Purification

Antigen Affinity-purified

Reconstitution

Reconstitute at 0.2 mg/mL in sterile PBS. For liquid material, refer to CoA for concentration.

Formulation

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.

Shipping

Lyophilized product is shipped at ambient temperature. Liquid small pack size (-SP) is shipped with polar packs. Upon receipt, store immediately at the temperature recommended below.

Stability & 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.

Calculators

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: STAT3

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.

Long Name

Signal Transducer and Activator of Transcription 3

Alternate Names

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

Entrez Gene IDs

6774 (Human); 20848 (Mouse); 25125 (Rat)

Gene Symbol

STAT3

Additional STAT3 Products

Product Documents for Human Phospho-STAT3 (Y705) Antibody

Certificate of Analysis

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Product Specific Notices for Human Phospho-STAT3 (Y705) Antibody

For research use only

Citations for Human Phospho-STAT3 (Y705) Antibody

Customer Reviews for Human Phospho-STAT3 (Y705) Antibody (3)

4.7 out of 5
3 Customer Ratings
5 Stars
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Showing  1 - 3 of 3 reviews Showing All
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  • Human Phospho-STAT3 (Y705) Antibody
    Name: Anonymous
    Application: Western Blot
    Sample Tested: HEK293 human embryonic kidney cell line
    Species: Human
    Verified Customer | Posted 07/21/2018
    Human Phospho-STAT3 (Y705) Antibody AF4607
  • Human Phospho-STAT3 (Y705) Antibody
    Name: Anonymous
    Application: Immunoprecipitation
    Sample Tested: IPS2 induced pluripotent stem cells
    Species: Human
    Verified Customer | Posted 01/17/2018
    Human Phospho-STAT3 (Y705) Antibody AF4607
  • Human Phospho-STAT3 (Y705) Antibody
    Name: Carme Caelles
    Application: Western Blot
    Sample Tested: Whole cell extracts from bone marrow derived macrophages
    Species: Mouse
    Verified Customer | Posted 04/05/2017
    Human Phospho-STAT3 (Y705) Antibody AF4607

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