Pser727 stat3

Manufactured by Cell Signaling Technology

Sourced in United States

PSer727-STAT3 is a laboratory reagent that detects phosphorylation of the serine 727 residue on the STAT3 protein. It is intended for use in research applications that require the identification and quantification of this specific post-translational modification.

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STAT3

6 protocols using pser727 stat3

Comprehensive Protein Expression Analysis

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Antibodies (Ab) against Chk1, Chk1-pSer296, Chk1-pSer317, Chk1-pSer345, p21, beta-Actin, H2AX, H2AX-pSer139, total PARP, cleaved-PARP, total caspase-3, cleaved-caspase-3, c-Myc, Bcl-2, Mcl-1, STAT3-pSer727, and STAT3-pTyr705 were purchased from Cell Signaling Technology. Anti-NFκB Ab was from Santa Cruz Biotechnology. Anti-p53 Ab (DO-1) was a gift from Dr. Borivoj Vojtesek (Masaryk Memorial Cancer Institute, Brno). Anti-rabbit and anti-mouse secondary antibodies were purchased from DakoCytomation. Anti-Ki-67-PE Ab and appropriate isotype control were purchased from BioLegend.

Zemanova J., Hylse O., Collakova J., Vesely P., Oltova A., Borsky M., Zaprazna K., Kasparkova M., Janovska P., Verner J., Kohoutek J., Dzimkova M., Bryja V., Jaskova Z., Brychtova Y., Paruch K, & Trbusek M. (2016). Chk1 inhibition significantly potentiates activity of nucleoside analogs in TP53-mutated B-lymphoid cells. Oncotarget, 7(38), 62091-62106.

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Protein Expression Analysis in Stem Cells

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Cultured cells were lysed in strong RIPA buffer containing Halt Protease Inhibitor Cocktails (Thermo, Waltham, MA, http:// www.thermo.com.cn/). The obtained protein concentrations were estimated using a BCA protein assay kit (Pierce, Rockford, IL, http://www.pierce-antibodies.com/). Primary antibodies targeting PWP1 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com), OCT4 (1:1,000, Santa Cruz Biotechnology), NANOG (1:1,000, Abcam, Cambridge, U.K., http://www.abcam.com), SOX2 (1:1,000, Abcam), H3K4me3 (1:1,000, Millipore), H3K9me3 (1:1,000, Millipore), H3K27me3 (1:1,000, Millipore), H4K20m3 (1:1,000, Millipore), STAT3 (1:1,000, Cell Signaling Technology, Boston, MA, http://www. cst-c.com.cn/), STAT3 (p-Ser727) (1:1,000, Cell Signaling Technology, Signalway, Baltimore, Maryland, http://www.sabbio-tech.com/), WNT3a (1:1,000, Cell Signaling Technology), SUV4-20H2 (1:1,000, Abcam), and GAPDH (1:1,000, Santa Cruz Biotechnology) were incubated with the proteins overnight at 4 C, followed by incubation with the appropriate HRP (horseradish peroxidase)-conjugated secondary antibodies. Detection of HRP was performed using the Super Signal West Pico Chemiluminescent Substrate (Pierce).

Shen J., Jia W., Yu Y., Chen J., Cao X., Du Y., Zhang X., Zhu S., Chen W., Xi J., Wei T., Wang G., Yuan D., Duan T., Jiang C, & Kang J. (2015). Pwp1 is required for the differentiation potential of mouse embryonic stem cells through regulating Stat3 signaling. Stem cells (Dayton, Ohio), 33(3).

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Antibody Immunoblot Analysis Protocol

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The antibodies for caspases-3, caspase-8, STAT3, pTyr705-STAT3, pSer727-STAT3, pSer70-bcl2, Bid, Bad, Akt, p38MAPK, pThr180/Tyr182-p38MAPK, cytochrome c, were from Cell Signaling Technology (Beverly, MA, USA). Antibodies for Bax and p21^CIP1 were from MBL (Nagoya, Japan). Antibodies for β-actin, Rb, Mcl-1, bcl-2, bcl-xL, p-gp130, JAK2, Cyclin D2, cyclin E, CDK2, CDK4, PKCα, PP2A/Aα, PP2A/B56α, PP2A/C were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for p-JAK2 or p27^kip1 were from Upstate (Waltham, MA, USA) or BD Transduction Laboratories, respectively. Anti-ERK1/2 or p-ERK1/2 antibody was from Sigma. Secondary antibodies conjugated with horseradish peroxidase were obtained from GE Healthcare (Tokyo, Japan).

SADAHIRA K., SAGAWA M., NAKAZATO T., UCHIDA H., IKEDA Y., OKAMOTO S., NAKAJIMA H, & KIZAKI M. (2014). Gossypol induces apoptosis in multiple myeloma cells by inhibition of interleukin-6 signaling and Bcl-2/Mcl-1 pathway. International Journal of Oncology, 45(6), 2778-2286.

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Quantitative Protein Analysis of Lung Tissues

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Total protein lysates were prepared from snap‐frozen lung tissues or cell lysates and subjected to ELISA and immunoblotting. Human and mouse IL‐6R ELISA sets were purchased from R&D Systems. Immunoblotting was performed with the following antibodies: total ADAM17 (from S. Rose‐John), pThr⁷³⁵‐ADAM17 (Sigma), Myc, pTyr⁷⁰⁵‐STAT3, pSer⁷²⁷‐STAT3, total STAT3, pSer⁴⁷³‐AKT, total AKT, pThr²⁰²/pTyr²⁰⁴‐ERK1/2, total ERK1/2, pThr¹⁸⁰/pTyr¹⁸²‐p38 MAPK, total p38 MAPK, pTyr¹⁰⁶⁸‐EGFR, total EGFR, pSer⁸²‐HSP27 (Cell Signaling Technology), cleaved Notch1 (Abcam), IL‐6R, Nrg1, pTyr¹²⁸⁹‐ErbB3, total ErbB3, TGFα (Santa Cruz Biotechnology), and actin (Sigma). Protein bands were visualized using the Odyssey Infrared Imaging System (LI‐COR) and quantified using Image J. Antibody dilutions are indicated in Appendix Table S2.

Saad M.I., Alhayyani S., McLeod L., Yu L., Alanazi M., Deswaerte V., Tang K., Jarde T., Smith J.A., Prodanovic Z., Tate M.D., Balic J.J., Watkins D.N., Cain J.E., Bozinovski S., Algar E., Kohmoto T., Ebi H., Ferlin W., Garbers C., Ruwanpura S., Sagi I., Rose‐John S, & Jenkins B.J. (2019). ADAM17 selectively activates the IL‐6 trans‐signaling/ERK MAPK axis in KRAS‐addicted lung cancer. EMBO Molecular Medicine, 11(4), e9976.

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Liver Signaling Pathway Analysis

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Liver tissues were homogenized in ice-cold NP40-Buffer containing 50 mM Tri-HCl (pH 7.5), 150 mM NaCl, 0.5% NP40 and 50 mM NaF freshly supplemented with Complete Mini (Roche), PhosSTOP (Roche), 1 mM orthovanadate and 1 mM pefablock. Protein concentrations were determined by BIO-RAD protein assay (BIO-RAD Laboratories GmbH, Munich, Germany). Samples were separated by SDS-PAGE and transferred to a cellulose membrane and probed with antibodies for pAKT, pERK, pTyr705 STAT3, pSer727 STAT3, pSTAT5, pJNK and pSMAD2/3 (Cell Signaling, Danvers, MA, USA). As secondary antibodies, anti-rabbit-HRP (Cell Signaling) and antimouse-HRP (Santa Cruz, Heidelberg, Germany) were used. GAPDH from AbD SeroTec (Düsseldorf, Germany) was used as loading control. Analysis of intrahepatic IL-6 (Abcam, Cambridge, UK) and OSM (R&D Systems, Minneapolis, MN) was performed in duplicates (n=6) using a murine ELISA kit.

Hatting M., Spannbauer M., Peng J., Al Masaoudi M., Sellge G., Nevzorova Y.A., Gassler N., Liedtke C., Cubero F.J, & Trautwein C. (2015). Lack of gp130 expression in hepatocytes attenuates tumor progression in the DEN model. Cell Death & Disease, 6(3), e1667-.

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Immunoblotting of Hepatic Signaling

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Western blots for immunocomplexes and hepatic homogenates were performed using antibodies against phosphorylated (p) Tyr705-signal transducer and activator of transcription factor 3 (pTyr705-STAT3), pSer727-STAT3 and SOCS3 from Cell Signaling Technology (Danvers, MA), pTyr1007/1008 Janus kinase 2 (pJAK2), JAK2 and regulatory subunit of PI3K (p85) from Millipore (Temecula, CA), STAT3 from R&D Systems (Minneapolis, MN) and aquaglyceroporin-9 (AQP9), the beta chain of insulin receptor (IRb), catalytic subunit of PI3K (p110), GLUT2, long form of the leptin receptor (OBeRb), phosphoenolpyruvate carboxykinase (PEPCK) and SH-PTP1 from Santa Cruz Biotechnology (Santa Cruz, CA). The proteins were detected by chemiluminiscence using an ECL system. Quantification of the bands was carried out by densitometry using a Kodak Gel Logic 1500 Image Analysis system and Molecular Imaging software 4.0 (Rochester, NY, USA). AQP9, GLUT2, IRb, OB-Rb, p85, PEPCK, SOCS3 and SH-PTP1 were normalized with actin (Thermo Scientific, Fremont, CA), whereas pJAK2, pTyr705-STAT3 and p-Ser727-STAT3 were normalized with their respective total forms.

Burgos-Ramos E., Canelles S., Rodríguez A., Gómez-Ambrosi J., Frago L.M., Chowen J.A., Frühbeck G., Argente J, & Barrios V. (2015). Chronic central leptin infusion modulates the glycemia response to insulin administration in male rats through regulation of hepatic glucose metabolism. Molecular and cellular endocrinology, 415.

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