P stat3

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom, China, Germany, Macao

P-STAT3 is an antibody that specifically detects phosphorylated STAT3 protein. STAT3 (Signal Transducer and Activator of Transcription 3) is a transcription factor that plays a key role in cellular signaling pathways. Phosphorylation of STAT3 is a crucial step in its activation and regulation of target gene expression.

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935 protocols using p stat3

Histological and Immunological Analysis of Aortic Dissection

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For histological analyses, AD tissues were fixed in 4% paraformaldehyde, paraffin-embedded, and sliced into 5-µm-thick tissue sections with circumferential orientation. Sections of aortic tissue were stained with hematoxylin and eosin (H&E), and elastica van Gieson (EVG) staining.
Immunofluorescence staining of the aortic tissue was performed using antibodies for STAT3 phosphorylated at Tyr705 (P-STAT3, Cell Signaling Technologies #9145, Danvers, MA, USA) with a TSA labeling kit with AlexaFluor 488 tyramide (Thermo Fisher Scientific #T-20922, Waltham, MA, USA), NFκB (Abcam, #ab13594, Cambridge, UK) with Cy3-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories #111-115-144, West Grove, PA, USA), and TOPRO-3 (Thermo Fisher Scientific #T3605, Waltham, MA, USA) for nuclear staining. Immunohistochemical staining of AD tissue for P-STAT3 (Cell Signaling Technologies #9145), and neutrophils (neutrophil elastase, Dako #M0752, Santa Clara, CA, USA) were also performed using avidin-biotin complex staining kits (Vectastain #PK-4001 and #PK-4002, Vector Labs, Burlingame, CA, USA) according to the instructions from the manufacturer.

Yoshida S., Yamamoto M., Aoki H., Fukuda H., Akasu K., Takagi K., Shojima T., Fukumoto Y., Akashi H, & Tanaka H. (2019). STAT3 Activation Correlates with Adventitial Neutrophil Infiltration in Human Aortic Dissection. Annals of Vascular Diseases, 12(2), 187-193.

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Nuclear Protein Extraction and Western Blot Protocol

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Nuclear protein was extracted according to the manufacturer's instruction of the nuclear and cytoplasmic protein extraction kit (Beyotime). The protocol for western blot has been described in detail in our previous publication.^{56 (link)} Primary antibodies were used as follows: antibodies against SHP-1 and SHP-2 (Epitomics, Burlingame, CA, USA); STAT3, p-STAT3 (Tyr705), p-STAT3 (Ser727), p-p44/42 Erk1/2 (Thr202/Tyr204), cyclin D1, cyclin E1, survivin, p-SHP-2 (Tyr580), p-SHP-2 (Tyr542) and Histon H3 (Cell Signaling Technology); antibody against Ki67 (BD Bioscience); antibodies against TC-PTP, SHP-2, Mcl-1 (Proteintech, Wuhan, Hubei, China); GAPDH (Beyotime).

Lu L., Zhang S., Li C., Zhou C., Li D., Liu P., Huang M, & Shen X. (2017). Cryptotanshinone inhibits human glioma cell proliferation in vitro and in vivo through SHP-2-dependent inhibition of STAT3 activation. Cell Death & Disease, 8(5), e2767-.

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Immunoblotting Analysis of pSTAT3 Signaling

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Vastus lateralis muscles were sectioned with a Leica CM 1900 cryostat (Walldorf, Baden-Wurttemberg, Germany). Thirty 10 μm cryosections were lysed in 50 μL of RIPA buffer (0.1% SDS, 1% NP40, 0.5% sodium deoxycholate, 150 mM sodium chloride, and 50 mM TrisHCl pH 7.5) for 30 minutes on ice, with protease inhibitor cocktail (Complete, Roche, Mannheim, Germany) as well as phosphatase inhibitor cocktail (PhosStop, Roche, Mannheim, Germany). At the end of the incubation, the cell extracts were centrifuged for 10 minutes (12,000 g) at 4°C. The amount of protein was calculated using the Quick Start Bradford Protein Assay Kit 1 (Bio Rad Laboratories, Hercules, CA). Then 30 μg of protein in NuPAGE LDS Sample Buffer (Life Technologies, Grand Island, NY) and NuPAGE Sample Reducing Agent (Life Technologies, Grand Island, NY) was loaded to SDS-PAGE gel for immunoblotting analysis. The primary antibodies used were pSTAT3 (Y705, 1 : 1000; Cell Signaling Technology, Danvers, MA), pSTAT3 (S727, 1 : 1000; Cell Signaling Technology, Danvers, MA), and Total STAT3 (1 : 1000; Cell Signaling Technology, Danvers, MA). Bound antibodies were detected using ECL reagents. The results were normalized to GAPDH (1 : 5000; Millipore, Billerica, MA). Band intensity was evaluated by densitometry analysis, normalized to its total content, and reported as fold increase relative to respective control set as 1.

Guadagnin E., Narola J., Bönnemann C.G, & Chen Y.W. (2015). Tyrosine 705 Phosphorylation of STAT3 Is Associated with Phenotype Severity in TGFβ1 Transgenic Mice. BioMed Research International, 2015, 843743.

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Characterization of Ovarian Cancer Cell Lines

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Ovarian cancer cell line OVCAR5 cells were purchased from the National Cancer Institute’s cell line repository. SKOV3, cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and HeyA8 cells were received from the characterized cell line core at MD Anderson Cancer Center. Cells were cultured in DMEM media (GIBCO, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Invitrogen Life Technologies, Carlsbad, CA) and 1% penicillin/streptomycin (GIBCO). ID8 cells (a kind gift from Dr. Weiguo Cui) were cultured in Dulbecco’s modified Eagle’s medium (GIBCO) supplemented with 10% FBS, 1% penicillin/streptomycin, 1% insulin (5 g/mL), 5 g/mL transferrin, 5 ng/mL sodium selenite (1X ITS; Sigma Cat. No. I3146). We obtained, ERBB2 (#2165), p-ERBB2 (Tyr1248)(#2247), ERBB3 (#12708), p-ERBB3 (Tyr1289), IGF1R-β (#3027), GAPDH (#5174), mesothelin (#99966S), TGF-β (#3711), STAT3 (#9139), p-STAT3 (Tyr705)(#9145), p-STAT3 (Ser727)(#34911), JAK1 (#3344S), p-JAK1(Tyr1034/1035) (#74129S), JAK2 (3230S), p-JAK2 (Tyr1008) (#8082S) TYK2 (#14193S) and p-TYK2 (Tyr1054/1055) (#68790S) from Cell Signaling Technology (Danvers, MA), Furin (#sc-133142) was obtained from Santa Cruz Biotechnology. Recombinant Human Neuregulin β−1 (NRG1), was purchased from PeproTech, Cat# 100–03.

Chen C., Gupta P., Parashar D., Nair G.G., George J., Geethadevi A., Wang W., Tsaih S.W., Bradley W., Ramchandran R., Rader J.S., Chaluvally-Raghavan P, & Pradeep S. (2020). ERBB3-induced furin promotes the progression and metastasis of ovarian cancer via the IGF1R/STAT3 signaling axis. Oncogene, 39(14), 2921-2933.

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Comprehensive Western Blot Analysis

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Western blotting was performed as previously described [39 (link)]. Briefly, cells were treated with indicated compounds and then lysed by boiling. Lysates were immunoblotted with antibodies against STAT3 (9139; Cell Signaling Technology), p-STAT3 (Y705, 9145; Cell Signaling Technology), p-STAT3 (S727, 9134; Cell Signaling Technology), CMYC (ab32072; Abcam), Cyclin D1 (ab134175; Abcam), Survivin (2808; Cell Signaling Technology), MMP2 (4022; Cell Signaling Technology), BCL-XL (2764; Cell Signaling Technology), hemagglutinin (M20003; Abmart), FLAG (F1804; Sigma-Aldrich), EGFR (4267; Cell Signaling Technology), NF-κB (4764; Cell Signaling Technology), E-cadherin (14472; Cell Signaling Technology), N-cadherin (13116; Cell Signaling Technology), vimentin (CY5134; Abways) and GAPDH (ab181602; Abcam). The secondary antibody was conjugated with IRDye 680/800 (926-32221, 926-32210; Millennium Science).

Chen H., Bian A., Yang L.F., Yin X., Wang J., Ti C., Miao Y., Peng S., Xu S., Liu M., Qiu W.W, & Yi Z. (2021). Targeting STAT3 by a small molecule suppresses pancreatic cancer progression. Oncogene, 40(8), 1440-1457.

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Ivermectin's Effects on HCC Cell Lines

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Human HCC cell lines HuH6, Hep3B and SNU‐182 (Cell Bank of Shanghai Institute of Biological Science) were cultured in Iscove’s modified Dulbecco’s medium (IMDM, Life Technologies) supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified 5% CO₂ environment. These cell lines were authenticated using human 9‐Marker STR DNA profile analysis. The cells were seeded in multiple‐well plates at 70% density and treated with ivermectin (R&D Systems) at 2.5, 5, 10 and 20 μM for 8 to 72 h depending on assay types (indicated in figure legends). Equal volume of DMSO was used as control. p‐mTOR (S2448, #2971), mTOR (#2983), p‐STAT3 (T705, #9131), p‐STAT3 (S727, #9134), STAT3 (#9139), Mcl‐1 (#94296), Bcl‐xL (#2762), c‐Myc (#9402), E‐cadherin (#3195), Vimentin (#3932), Snail (#3879), Slug (#9858), Nanog (#3580), Sox‐2 (#2748), Oct‐4 (#2750) were purchased from Cell Signaling.

Lu H., Zhou L., Zuo H., Le W., Hu J., Zhang T., Li M, & Yuan Y. (2022). Ivermectin synergizes sorafenib in hepatocellular carcinoma via targeting multiple oncogenic pathways. Pharmacology Research & Perspectives, 10(3), e00954.

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Quantifying pSTAT3 in Human Gliomas

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To assess the degree of pSTAT3 staining in human gliomas, two separate tissue microarrays comprising formalin-fixed, paraffin embedded (FFPE)tissue cores of human low- and high-grade gliomas were stained with pSTAT3 (Tyr705; 0.2 μg/ml; Cell Signaling Technology, Boston MA) and counterstained with DAPI. Immunofluorescence experiments were performed using Discovery XT processor (Ventana Medical Systems, Oro Valley, AZ) at the Molecular Cytology Core Facility (MSKCC). The slides were scanned at high resolution and analyzed using Panoramic Viewer Software. Up to 3 tissue cores per case were scanned at low power for an area of maximal pSTAT3 staining. A representative 400X field from this area was selected for quantification. Distinct nuclear staining above background was considered positive. In each image, 100 to 500 cells were manually counted using Image J software's cell counter and grid macros. A two-tailed Student's t-test was used to assess for a significant difference in positive pSTAT3 staining between high-grade glioma (n=41) and low-grade glioma (n=38) samples (total n=79).Multiplex immunofluorescent staining for pSTAT3/GFP/CD11b was performed as previously described (28 (link)).

Rajappa P., Cobb W.S., Vartanian E., Huang Y., Daly L., Hoffman C., Zhang J., Shen B., Yanowitch R., Garg K., Cisse B., Haddock S., Huse J., Pisapia D.J., Chan T.A., Lyden D.C., Bromberg J.F, & Greenfield. J.P. (2016). Malignant astrocytic tumor progression potentiated by JAK-mediated recruitment of myeloid cells. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(12), 3109-3119.

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Characterization of Ovarian Cancer Cell Lines

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Multimodal Protein Visualization in Biological Samples

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The sections were incubated overnight at room temperature using standard procedures for the primary and secondary antibodies below. The following day, sections were washed and incubated with either biotinylated antibodies (1:200, Jackson Immunoresearch, 711-065-152) followed by avidin-biotin complex amplification and 3,3-diaminobenzidine (Thermo Fisher Scientific, 32020 and 34065) or fluorescent secondary antibodies with species-specific Alexa Fluor 488 or 568 (1:250, Invitrogen, A-11039 or A-11011) to visualize proteins. Primary antibodies used include GFP (1:1,000, Aves Laboratories, 1020), FOS (1:1,000, Cell Signaling, 2250), dsRed (1:1,000, Takara Bio, 632496), POMC (1:1,000, Phoenix Pharmaceuticals Inc., H-029-30), and pSTAT3 (1:500, Cell Signaling, 9145). Images were collected on an Olympus BX51 or Olympus BX53 microscope. Images were background subtracted and enhanced by shrinking the range of brightness and contrast in ImageJ (NIH).

Rupp A.C., Tomlinson A.J., Affinati A.H., Yacawych W.T., Duensing A.M., True C., Lindsley S.R., Kirigiti M.A., MacKenzie A., Polex-Wolf J., Li C., Knudsen L.B., Seeley R.J., Olson D.P., Kievit P, & Myers MG J.r. (2023). Suppression of food intake by Glp1r/Lepr-coexpressing neurons prevents obesity in mouse models. The Journal of Clinical Investigation, 133(19), e157515.

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Macrophage Polarization Pathway Analysis

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Phorbol-12-myristate-13-acetate (PMA) was bought from Sigma Aldrich (St. Louis, Missouri, USA), human IL-4 and human IL-13 were purchased from Peprotech (200-04 and 200-13, Rocky Hill, USA).The anti-CD163, anti-CD206, anti-β2-AR and anti-PKA antibodies were purchased from Santa Cruz Biotechnology(sc-33559, sc-58986, sc-271322 and sc-98951, Ca, USA). Phenylmethylsulfonyl fluoride (PMSF) was bought from beyotime biotechnology (Shanghai, China). Primary antibodies against STAT3, pSTAT3, pCREB were purchased from Cell Signaling Technology (4904s, 9131s and 9198s, Danvers, MA, USA). Anti-CREB antibody was purchased from Abcam (ab32515, Cambridge, MA, USA). AlexaFluor488 donkey anti-mouse antibody and AlexaFluor594 goat anti-rabbit antibody were purchased from ThermoFisher Scientific (A11037, A21202, Waltham, MA, USA). Fluorescein isothiocyanate dextran (FITC-dextran) was from Sigma Aldrich (FD40s, St. Louis, Missouri, USA). AlexFluor 647 was from Fcmacs (FMS-Msaf64701, Nanjing, Jiangsu, China).

Wu J.J., Yang Y., Peng W.T., Sun J.C., Sun W.Y, & Wei W. (2019). G protein-coupled receptor kinase 2 regulating β2-adrenergic receptor signaling in M2-polarized macrophages contributes to hepatocellular carcinoma progression. OncoTargets and therapy, 12, 5499-5513.

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