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P stat3 d3a7

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P-STAT3 (D3A7) is a rabbit monoclonal antibody that detects phosphorylated STAT3 (Tyr705). STAT3 is a transcription factor that becomes phosphorylated in response to various cytokines and growth factors.

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Lab products found in correlation

Ripa buffer, by Merck Group (2 mentions) Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (2 mentions) Hif 1α, by Cayman Chemical (2 mentions) Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions) Chemidoc imager, by Bio-Rad (2 mentions) Pten d4.3, by Cell Signaling Technology (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) E cadherin, by Cell Signaling Technology (1 mentions) Snail c15d3, by Cell Signaling Technology (1 mentions) Mmp9 d6o3h, by Cell Signaling Technology (1 mentions) Stat3 d3z2g, by Cell Signaling Technology (1 mentions) Mmp 2, by Cell Signaling Technology (1 mentions) Vimentin, by Cell Signaling Technology (1 mentions) Anti actin, by Thermo Fisher Scientific (1 mentions) Anti p akt, by Cell Signaling Technology (1 mentions) P stat1, by Cell Signaling Technology (1 mentions) Anti akt, by Abcam (1 mentions) Anti cd86, by Abcam (1 mentions) Anti p65, by Santa Cruz Biotechnology (1 mentions) Anti p p65, by Santa Cruz Biotechnology (1 mentions)

Spelling variants of this product

STAT3 (1075 citations) P-STAT3 (935 citations) Rabbit anti–p-STAT3 (Tyr705) (clone D3A7, (2 citations)

12 protocols using p stat3 d3a7

1

Immunoblotting Analysis of EMT Markers

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Western blotting was performed as previously described37 (link). The antibodies used in this study were E-cadherin (24E10, Cell Signaling Technology, USA), MMP9 (D6O3H, Cell Signaling Technology, USA), MMP2 (D4M2N Cell Signaling Technology, USA), vimentin (D21H3, Cell Signaling Technology, USA), PTEN (D4.3, Cell Signaling Technology, USA), p-PTEN (44A7, Cell Signaling Technology, USA), STAT3 (D3Z2G, Cell Signaling Technology, USA), p-STAT3 (D3A7, Cell Signaling Technology, USA), snail (C15D3, Cell Signaling Technology, USA), and GAPDH (D16H11, Cell Signaling Technology, USA). GAPDH was used as an internal control. The immunoreactive bands were visualized with ECL Ultra (New Cell and Molecular Biotech, Suzhou, China). All western blots were repeated three times with separate cell lysates and the statistical analysis were provided in the supplementary figures.
Xu Q., Ma H., Chang H., Feng Z., Zhang C, & Yang X. (2020). The interaction of interleukin-8 and PTEN inactivation promotes the malignant progression of head and neck squamous cell carcinoma via the STAT3 pathway. Cell Death & Disease, 11(5), 405.
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2

Comprehensive Immunohistochemical Antibody Panel

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The following primary antibodies were used: anti-p65 (sc-8008), anti-pp65 (sc-166748) anti-IκBα (sc-1643), anti-MUC1 VU-4H5 (sc-7313, Santa Cruz), anti-ST6GALNAC1 (PA5-31200 and 15363-1-AP), anti-IL-13 (AHC0132), anti-actin (MA5-11869, Thermo Fisher Scientific), anti-MUC1 5E5 (TAB-418MZ, Creative Biolabs), anti-CD163 (NBP2-36494, Novus Biologicals), anti-CCL17 (ab182793), anti-sialyl Tn, clone sTn 219 (ab115957), anti-CD68 (ab955), anti-CD86 (ab53004, Abcam), anti-AKT (C67E7, #4691), anti-p-AKT (D9E, #4060), p-STAT1 (D4A7, #7649), p-STAT3 (D3A7, #9145), p-STAT6 (D8S9Y, #56554, Cell Signaling).
Kvorjak M., Ahmed Y., Miller M.L., Sriram R., Coronello C., Hashash J.G., Hartman D.J., Telmer C.A., Miskov-Zivanov N., Finn O.J, & Cascio S. (2019). Crosstalk between colon cells and macrophages increases ST6GALNAC1 and MUC1-sTn expression in ulcerative colitis and colitis–associated colon cancer. Cancer immunology research, 8(2), 167-178.
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3

FISH Analysis of 9p24.1 in Hodgkin's Lymphoma

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Fluorescence in situ hybridization (FISH) was performed on Hodgkin's lymphoma tissue sections to assess copy number on chromosome 9p24.1. The bacterial artificial chromosome probes (CHORI; www.chori.org) RP11-599H2O, which maps to 9p24.1 and includes CD274 (encoding PD-L1, labeled with Spectrum Orange), and RP11-635N21, which also maps to 9p24.1 and includes PDCD1LG2 (encoding PD-L2, labeled with Spectrum Green), were cohybridized. A control centromeric probe, Spectrum Aqua–labeled CEP9 (Abbott Molecular) that maps to 9p11-q11, was hybridized according to the manufacturer's recommendations. Malignant Reed–Sternberg cells were identified by means of nuclear morphologic features, and all such cells were analyzed. Nuclei with a target:control probe ratio of at least 3:1 were classified as amplified, those with a probe ratio of more than 1:1 but less than 3:1 were classified as relative copy gain, and those with a probe ratio of 1:1 but with more than two copies of each probe were classified as polysomic for chromosome 9p. Immunohistochemical staining was performed by means of an automated staining system (BOND-III, Leica Biosystems), with the use of a double-staining technique for PD-L1 (405.9A11) and PAX5 (24/Pax-5, BD Biosciences), and for PD-L2 (366C.9E5) and phosphorylated STAT3 (pSTAT3; D3A7, Cell Signaling Technology). The methods are detailed in the Supplementary Appendix.
Ansell S.M., Lesokhin A.M., Borrello I., Halwani A., Scott E.C., Gutierrez M., Schuster S.J., Millenson M.M., Cattry D., Freeman G.J., Rodig S.J., Chapuy B., Ligon A.H., Zhu L., Grosso J.F., Kim S.Y., Timmerman J.M., Shipp M.A, & Armand P. (2014). PD-1 Blockade with Nivolumab in Relapsed or Refractory Hodgkin's Lymphoma. The New England journal of medicine, 372(4), 311-319.
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4

Western Blot Analysis of Phospho-STAT3

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Cells were lysed in RIPA buffer supplemented with protease inhibitor cocktail (PIC, 1x), sodium fluoride (NaF, 200 mM), phenylmethylsulfonyl fluoride (PMSF, 1 mM), ethylenediaminetetraacetic acid (EDTA, 5 mM) and calyculin A (50 nM, Cell Signaling, USA). For the preparation of protein samples from CM, PIC (1x) and EDTA (5 mM) were added into the cell culture supernatant fraction. Following the quantitation with colorimetric bicinchoninic acid (BCA) assay, total protein (50–120 μg) was subjected to SDS-PAGE on 10% gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% skimmed milk powder in PBS-T buffer and incubated overnight at 4 °C with primary antibodies reactive with human phospho-STAT3 Tyr705 (pSTAT3; D3A7, 1/1500), total STAT3 (tSTAT3; 79D7, 1/1500), and β-actin (D6A8, 1/5000) (Cell Signaling), fibronectin (10/Fibronectin, 1/1000) (BD), FN-EDA (IST-9, 1/1000) (Abcam, UK). Following the incubation with HRP-conjugated secondary antibodies (goat anti-rabbit IgG, 1/3000; horse anti-mouse IgG, 1/1500) (Cell Signaling) (1.5 h, at room temperature), and then, with ECL substrate solution, protein bands were visualized and densitometric analyses were performed (Kodak Gel Logic 1500, Carestream, USA). Otherwise stated, all materials were purchased from Thermo Scientific, USA.
Tunali G., Yanik H., Ozturk S.C., Demirkol-Canli S., Efthymiou G., Yilmaz K.B., Van Obberghen-Schilling E, & Esendagli G. (2023). A positive feedback loop driven by fibronectin and IL-1β sustains the inflammatory microenvironment in breast cancer. Breast Cancer Research : BCR, 25, 27.
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5

Antibody and Inhibitor Characterization

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The GAPDH (6C5) and β‐actin (C4) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The anti‐human ERα (D8H8), ERK1/2 (137F5), p‐ERK1/2 (197G2), AKT (11E7), p‐AKT (244F9), and p‐STAT3 (D3A7) antibodies for western blot were purchased from Cell Signaling Technology (Boston, MA, USA); MMP‐9 (ab38898) antibody was from Abcam (Cambridge, MA, USA). The anti‐mouse ERα (E115) for western blot was from Abcam. The CXCL12 (AF‐310‐NA) antibody for western blot was from R&D Systems (Minneapolis, MN, USA). Anti‐mouse/anti‐rabbit secondary antibody for western blot was from Invitrogen (Carlsbad, CA, USA). Normal rabbit IgG was also from Santa Cruz Biotechnology. MPP (CAS 911295‐24‐4) was from Bio‐Techne Corporation (Minneapolis, MN, USA). PMA (CAS 16561‐29‐8) and CCR2 antagonist (CAS 445479‐97‐0) were from MilliporeSigma (Burlington, MA, USA). AMD3100 (CAS 155148‐31‐5) was from Bio‐Techne Corporation. U0126 (CAS 109511‐58‐2) was from Cell Signaling Technology. MK‐2206 (CAS 1032350‐13‐2) and fulvestrant (CAS 129453‐61‐8) were from Selleckchem (Houston, TX, USA).
He M., Yu W., Chang C., Miyamoto H., Liu X., Jiang K, & Yeh S. (2020). Estrogen receptor α promotes lung cancer cell invasion via increase of and cross‐talk with infiltrated macrophages through the CCL2/CCR2/MMP9 and CXCL12/CXCR4 signaling pathways. Molecular Oncology, 14(8), 1779-1799.
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6

Western Blot Analysis of Hypoxia Signaling Pathway

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Cells were lysed in chilled 1x RIPA buffer (10x stock, EMD Millipore) diluted in PBS containing 1 tablet of cOmplete EDTA free pro- tease inhibitor and PhosSTOP phosphatase inhibitor for 30 minutes on ice. Protein was quantified using a Pierce BCA protein quantification kit. 20 μg of total protein lysates were loaded and separated on NuPAGE 4–12% Bis-Tris gels (Thermo Scientific). Proteins were transferred to nitrocellulose membranes, blocked for 1 hour with 5% milk in TBS-T, and incubated with the indicated primary antibodies diluted in TBS-T at 4°C overnight. Membranes were washed in TBS-T and incubated in HRP-coupled secondary anti- bodies at room temperature. Proteins were detected by chemiluminescence using ECL (Thermo Scientific) in a Bio-Rad ChemiDoc Imager. The following primary antibodies and dilutions were used: vinculin (Sigma, V9131 1:2000), HIF-1α (Cayman Chemical, 10006421, 1:1000), STAT3 (124H6, Cell Signaling, 1:1000), p-STAT3 (D3A7, Cell Signaling 1:1000), LDHA (21799–1-AP, Proteintech Group, 1:5000) and GLUT1 (ab115730 Abcam 1:1000). Western blot images were processed using Adobe Photoshop CS5.
Cutler RE J.r., Stephens R.M., Saracino M.R, & Morrison D.K. (1998). Autoregulation of the Raf-1 serine/threonine kinase. Proceedings of the National Academy of Sciences of the United States of America, 95(16), 9214-9219.
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7

Immunoblotting of PD-1, STAT3, and p-STAT3

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BMDCs in respective experiments were lysed in lysis buffer A (150 mM NaCl, 20 mM Tris Cl (pH7.4), 5 mM EDTA, 1 mM CaCl2, 10 mM NaF, 1% Triton X 100, 5% glycerol, HALT protease/phosphatase inhibitor (Thermo Scientific). 10 μgs protein were loaded and separated by SDS-PAGE and analyzed by immunoblotting for PD-1 (Novus Biologicals, AF1021), STAT3 (79D7) and p-STAT3 (D3A7) (Cell Signaling Technology), and β-actin (LI-COR, Lincoln NE). Corresponding anti-goat, anti-mouse, and anti-rabbit secondary antibodies conjugated to IRDyes were purchased from LI-COR. Signal was detected using LI-COR Odyssey Imaging System.
Lamichhane P., Karyampudi L., Shreeder B., Krempski J., Bahr D., Daum J., Kalli K.R., Goode E.L., Block M.S., Cannon M.J, & Knutson K.L. (2017). IL-10 release upon PD-1 blockade sustains immunosuppression in ovarian cancer. Cancer research, 77(23), 6667-6678.
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8

Immunohistochemical Analysis of Lung Tumor Markers

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Lungs were fixed in 10% formalin, paraffin embedded, sliced and mounted on slides. Slides were stained with hematoxylin-eosin (H&E). Sections were evaluated for Ki67, P-MAPK, P-STAT3, F4/80, IL-17A, Sox-2, P-NFκB and VEGF expression in bronchial epithelium, preneoplastic or tumor areas. Sections were incubated with primary antibodies overnight in a humidified chamber at 4°C with the following antibodies: Ki67 (ab833; 1:50; Abcam), P-MAPK (4370; 1:400; Cell Signaling Technology), P-STAT3 (D3A7; 1:400; Cell Signaling Technology), F4/80 (MCA497R; 1:100; Serotec), IL-17A (ab79056; 1:500; Abcam), Sox-2 (ab97959; 1:1000; Abcam), P-NFκB (ab86299; 1:200; Abcam) or VEGF (ab52917; 1:200; Abcam). Positive and negative controls were included for each marker. Bright field images were captured using a Leica DMI 3000B scope and LASv4.7 software (Leica Biosystems). IL-17A, Sox-2, P-NFκB and VEGF staining was graded as low (<30% positive cells per field), moderate (30-60% positive cells per field) or high (>60% positive cells per field). Ki67 cell proliferation index quantitated as the number of positive cells per field divided by the total number of tumor cells per field. For P-MAPK and P-STAT3 quantitation, number of positive cells per field were counted. Tumor infiltrating macrophages (F4/80 staining) was quantitated as the number of positive cells per field.
Stabile L.P., Farooqui M., Kanterewicz B., Abberbock S., Kurland B.F., Diergaarde B, & Siegfried J.M. (2017). Preclinical Evidence for Combined Use of Aromatase Inhibitors and NSAIDs as Preventive Agents of Tobacco-Induced Lung Cancer. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer, 13(3), 399-412.
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9

Immunoblotting Analysis of STAT3 and Arg-1

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The immunoblotting technique was performed as previously described (32 (link)). Equal amounts of cellular protein were separated by SDS-PAGE on a 10% gel, and immunoblotted with the following antibodies: β-actin (AC-15; Boster Biological Technology), t-STAT3 (124H6; Cell Signaling Technology), p-STAT3 (D3A7; Cell Signaling Technology), and Arg-1 (D4E3M; Cell Signaling Technology). Immunoblots were visualized using an ECL kit (Thermo Fisher Scientific). Western blotting results were quantified using the Image J software (NIH, Bethesda, MD, USA).
Tao H., Liu M., Wang Y., Luo S., Xu Y., Ye B., Zheng L., Meng K, & Li L. (2021). Icaritin Induces Anti-tumor Immune Responses in Hepatocellular Carcinoma by Inhibiting Splenic Myeloid-Derived Suppressor Cell Generation. Frontiers in Immunology, 12, 609295.
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10

Western Blot Analysis of Hypoxia Signaling Pathway

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Cells were lysed in chilled 1x RIPA buffer (10x stock, EMD Millipore) diluted in PBS containing 1 tablet of cOmplete EDTA free pro- tease inhibitor and PhosSTOP phosphatase inhibitor for 30 minutes on ice. Protein was quantified using a Pierce BCA protein quantification kit. 20 μg of total protein lysates were loaded and separated on NuPAGE 4–12% Bis-Tris gels (Thermo Scientific). Proteins were transferred to nitrocellulose membranes, blocked for 1 hour with 5% milk in TBS-T, and incubated with the indicated primary antibodies diluted in TBS-T at 4°C overnight. Membranes were washed in TBS-T and incubated in HRP-coupled secondary anti- bodies at room temperature. Proteins were detected by chemiluminescence using ECL (Thermo Scientific) in a Bio-Rad ChemiDoc Imager. The following primary antibodies and dilutions were used: vinculin (Sigma, V9131 1:2000), HIF-1α (Cayman Chemical, 10006421, 1:1000), STAT3 (124H6, Cell Signaling, 1:1000), p-STAT3 (D3A7, Cell Signaling 1:1000), LDHA (21799–1-AP, Proteintech Group, 1:5000) and GLUT1 (ab115730 Abcam 1:1000). Western blot images were processed using Adobe Photoshop CS5.
Liu S., Hur Y.H., Cai X., Cong Q., Yang Y., Xu C., Bilate A.M., Gonzales K.A., Parigi S.M., Cowley C.J., Hurwitz B., Luo J.D., Tseng T., Gur-Cohen S., Sribour M., Omelchenko T., Levorse J., Pasolli H.A., Thompson C.B., Mucida D, & Fuchs E. (2023). A tissue injury sensing and repair pathway distinct from host pathogen defense. Cell, 186(10), 2127-2143.e22.
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