Phospho stat3 s727

Manufactured by Cell Signaling Technology

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Phospho-STAT3 S727 is a lab equipment product that detects phosphorylation of STAT3 at serine 727. This phosphorylation event plays a role in the regulation of STAT3 transcriptional activity.

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6 protocols using phospho stat3 s727

STAT3 ChIP Assay in Crohn's Disease

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ChIP assay were performed using nuclear extracts from freshly isolated smooth muscle cells of non-CD subjects, and from patients with stricturing Crohn's disease. In other experiments primary cultured muscle cells were was used after transfection with various STAT3 mutants and treatment of cells with IL-6 (10ng/ml) for 6 hours prior to extraction of genomic DNA using Trizol (Invitrogen, Carlsbad, CA) for quantitative analysis. ChIP-grade antibodies against total STAT3, phospho-STAT3(S727) or phospho-STAT3(Y705) (Cell Signaling, Boston, MA) were used in addition to control rabbit IgG. PCR was performed with primers specific for the promoter region of TGFB1 gene (Switchgear, Menlo Park, CA). Results were calculated from input, which is referred to PCR without immunoprecipitation:

% Input (normalized) = % Input (STAT 3) - % Input (Control IgG) .

Li C., Iness A., Yoon J., Grider J.R., Murthy K.S., Kellum J.M, & Kuemmerle J.F. (2015). Non-canonical STAT3 activation regulates excess TGF-β1 and Collagen I expression in muscle of stricturing Crohn's disease. Journal of immunology (Baltimore, Md. : 1950), 194(7), 3422-3431.

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Raddeanin A-induced Apoptosis Mechanisms

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Raddeanin A was purchased from Shanghai Yuanye Biotechnology, Ltd (Shanghai, China). Stock solution (20 mmol/L) was prepared by dissolution in DMSO and stored in the dark at −20°C. GSH (glutathione, γ‐glutamyl cysteinyl glycine), cycloheximide (CHX), SP600125 (JNK inhibitor), and NSC74859 (STAT3 inhibitor, S3I‐201) were purchased from Sigma‐Aldrich (St Louis, MO, USA). LY3214996 (ERK1/2 inhibitor) and Q‐VD‐OPh (caspase inhibitor) were purchased from MCE (Monmouth Junction, NJ, USA). Antibodies against STAT3, phospho‐STAT3^S727, Bcl‐2, Bcl‐xl, Bax, total‐ and cleaved PARP, cleaved caspase‐3, cleaved caspase‐8, cleaved caspase‐9, c‐Jun, phospho‐c‐Jun, phospho‐JNK, JNK, ERK1/2, phospho‐ERK1/2 and actin were purchased from Cell Signaling Technology (Beverly, MA, USA).

Wang Z., Shen J., Sun W., Zhang T., Zuo D., Wang H., Wang G., Xu J., Yin F., Mao M., Zhou Z., Hua Y, & Cai Z. (2019). Antitumor activity of Raddeanin A is mediated by Jun amino‐terminal kinase activation and signal transducer and activator of transcription 3 inhibition in human osteosarcoma. Cancer Science, 110(5), 1746-1759.

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Western Blot Analysis of mTOR Pathway

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Cell extracts were prepared by scraping the cells with 60–80 μL of cold lysis buffer (Lee et al., 2014) and storing on ice for 10 min. Clearing of the homogenates, protein quantification, SDS/PAGE, western blotting, and quantification of blots were performed as described previously (Lee et al., 2009). The antibodies (Catalog number) against 4E‐BP1 (#9644), phospho‐4E‐BP1(T37/46) (#2855), phospho‐4E‐BP1(S65) (#9451), phospho‐4E‐BP1(T70) (#9455), 4E‐BP2 (#2845), AKT (#4691), phospho‐AKT(T308) (#2965), phospho‐AKT(S473) (#9271), eIF4A (#2013), eIF4A1 (#2490), eIF4B (#3592), phospho‐eIF4B(S422) (#3591), eIF4E (#2067), eIF4G (#2469), eIF4H (#3469), mLST8 (#3274), Raptor (#2280), Rictor (#2214), p70S6K (#2708), phospho‐p70S6K (T389) (#9205), STAT1 (#9172), phospho‐STAT1(Y701) (#9171), STAT3 (#4904), phospho‐STAT3(Y705) (#9131), phospho‐STAT3(S727) (#9134), mTOR (#2983), phospho‐mTOR(S2448) (#5536), and phospho‐mTOR(S2481) (#2974) were obtained from Cell Signal Technology (Danvers, MA, USA); GAPDH (#CSB‐MA000071M0m) was obtained from Cusabio (Houston, TX, USA).

Lee H., Chin H., Kim H., Jung H, & Lee D. (2020). STAT3‐mediated MLST8 gene expression regulates cap‐dependent translation in cancer cells. Molecular Oncology, 14(8), 1850-1867.

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Tissue Protein Expression Analysis

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N-HLF, IPF-HLF or 10 mg of lung tissue samples were lysed, and Western blot was performed as previously described [26 (link)]. For phospho/total (p/t) Smad3, p/t Stat3 and suppressors of cytokine signaling 3 (SOCS3) expression levels, proteins were extracted directly from the tissue sample (10 mg, following biopsy) and from the cell line culture flasks during regular cell passages (between passages 3–10, during the normal proliferation phase). The following rabbit/mouse anti-human antibodies were used: phospho-Stat3 Tyr705 (#9145), phospho-Stat3 S727 (#9134), Stat3 (#9139), phospho-Smad3 (#9520) and Smad3 (#9523) from Cell Signaling Technologies, USA; Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) (ab9484) and SOCS3 (ab16030) were purchased from Abcam, USA; alpha-Tubulin (T5168) was purchased from Sigma, USA. Bound antibodies were visualized using Goat peroxidase-conjugated secondary antibodies (Millipore, USA, anti-Mouse IgG #AP308P and anti-Rabbit IgG #AP307P) followed by enhanced chemiluminescence detection (Millipore, USA). Results were normalized to Tubulin and GAPDH.

Epstein Shochet G., Brook E., Bardenstein-Wald B, & Shitrit D. (2020). TGF-β pathway activation by idiopathic pulmonary fibrosis (IPF) fibroblast derived soluble factors is mediated by IL-6 trans-signaling. Respiratory Research, 21, 56.

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Evaluating Cancer Cell Signaling Pathways

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The cisplatin modified DNA (ab103261) and γH2A.X (ab26350) antibodies were from Abcam (Cambridge, USA). The phospho-STAT3 S727 (#9134), phospho-Akt S473 (#9271), phospho-NF-κB S536 (#3033), pERK T202/Y204 (#9101), pATF2 T71 (#5112), (P70S6K (#2708), phospho-P70S6K T389 (#9205), p21 (#2947), cleaved caspase 3 (#9661) and cleaved caspase 7 (#9491) antibodies were from Cell Signaling (MA, United States). The γH2A.X (S139) antibody (AB26350) was from Abcam (MA, USA). The p53 antibody (sc-126) was from Santa Cruz Biotechnology (TX, USA). The p63 antibody (NB100-691) was from Novus Biologicals (CO, USA). The actin monoclonal antibody (AC-15) was from Sigma-Aldrich (MO, United States). The SignalSilence p70/85 S6 Kinase siRNA was from Cell Signaling (MA, United States). The plasmids for FUCCI live cell imaging, mVenus-hGeminin(1/110) and mCherry-hCdt1(30/120), were a kind gift from Dr Atsushi Miyawaki (Riken, Japan). Dactolisib (NZP-BEZ235), MK2206, S3I-201, UO126 and SC75741 were all from Selleck Chem (MA, USA).

Hastings J.F., Gonzalez Rajal A., Latham S.L., Han J.Z., McCloy R.A., O'Donnell Y.E., Phimmachanh M., Murphy A.D., Nagrial A., Daneshvar D., Chin V., Watkins D.N., Burgess A, & Croucher D.R. (2020). Analysis of pulsed cisplatin signalling dynamics identifies effectors of resistance in lung adenocarcinoma. eLife, 9, e53367.

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Western Blot Analysis of Signaling Proteins

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Cells were starved overnight, treated under the desired conditions, and lysed with RIPA buffer (Sigma-Aldrich) supplemented with phosphatase and protease inhibitors (Roche, Basel, Switzerland). Whole-cell lysates were subjected to acrylamide SDS-PAGE using standard procedures, transferred onto a nitrocellulose support membrane (GE Healthcare, Chicago, IL), and western blotted. The following antibodies were used: a Tubulin (Santa Cruz Biotechnology, Dallas, TX); FLAG (DDK, Origene Technologies); phospho-STAT3 Y705, phospho-STAT3 S727, STAT3, and protein kinase C q (Cell Signaling, Danvers, MA); and goat anti-mouse IgG DyLight 800 and goat anti-rabbit IgG Dylight 680 (Invitrogen). Bands were visualized and recorded with an Odyssey Infrared Imaging scanner (LI-COR Biosciences, Lincoln, NE) and were quantified by densitometry using Image Studio Software (LI-COR Biosciences).

García-Díaz N., Casar B., Alonso-Alonso R., Quevedo L., Rodríguez M., Ruso-Julve F., Esteve-Codina A., Gut M., Gru A.A., González-Vela M.C., Gut I., Rodriguez-Peralto J.L., Varela I., Ortiz-Romero P.L., Piris M.A, & Vaqué J.P. (2022). PLCγ1/PKCθ Downstream Signaling Controls Cutaneous T-Cell Lymphoma Development and Progression. The Journal of investigative dermatology, 142(5).

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