Phospho-STAT3 (Ser727) Polyclonal Antibody Catalog Number

Transcription

1 Website: thermofisher.com Customer Service (US): ext. 1 Technical Support (US): ext. 441 Phospho-STAT3 (Ser727) Polyclonal Antibody Catalog Number G Product data sheet Details Size Host/Isotope Class Type Immunogen Conjugate Form Purification Storage buffer Contains Storage Conditions 10 blots Rabbit / IgG Polyclonal Antibody Synthetic phosphopeptide from human STAT3 containing serine 727. This region is conserved between human, mouse and rat. Unconjugated Liquid Antigen affinity chromatography Dulbecco's PBS, ph 7.3, with 1mg/ml BSA, 50% glycerol 0.05% sodium azide -20 C Species Reactivity Tested species reactivity Published species reactivity Human, Mouse, Rat Tested Applications Dilution * ChIP assay (ChIP) 10µl Immunohistochemistry (Paraffin) (IHC (P)) Human, Mouse, Not Applicable 1:10-1:100 Western Blot (WB) 1:200-1:2000 Published Applications Immunofluorescence (IF) Western Blot (WB) Immunohistochemistry (IHC) Immunoprecipitation (IP) See 1 publications below See 8 publications below See 1 publications below See 1 publications below * Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls. Background/Target Information STATs (signal transducers and activators of transcription) were originally discovered as two proteins (STAT1 and STAT2) which were involved in interferonalpha (IFN-alpha) and IFN-gamma signal transduction. Since then, several additional STAT proteins have been identified (STAT3, 4, 5a, 5b, and 6). STATs undergo tyrosine phosphorylation in response to growth factor or cytokine signaling. This phosphorylation results in dimerization and translocation of STAT proteins to the nucleus. In some cases this process is mediated by JAK Kinases (Janus Kinases 1, 2, and 3). For maximum activation of these proteins, phosphorylation at specific tyrosine and serine residues may be required in STAT1 alpha, 3, 4, and 5. Specific functions of the various members of the STAT family are poorly understood. STAT3 has been shown to be activated by IFN-alpha but not IFN-beta. The transcription factors associated with STAT3 are c- Jun and cyclic AMP-responsive enhancer binding protein (CREB). Deletion of the STAT3 gene in knock-out mice was lethal at the early embryonic stage.

2 Advanced Verification Data Phospho-STAT3 (Ser727) Antibody (44-384G) in RE Phospho-STAT3 (Ser727) Antibody (44-384G) in TM Antibody specificity was demonstrated through detection of enrichment of the target protein at specific gene loci. Chromatin Immunoprecipitation (ChIP) was performed using anti-stat3 [ps727] Rabbit Polyclonal Antibody (Product # G) with relevant positive (GLS1)and negative target genes/ binding sites(cfos) Relative expression validation info. Altered expression of proteins upon cell treatment demonstrates antibody specificity. Western blot of Phospho-STAT3 (Ser727) using anti-stat3 [ps727] Rabbit Polyclonal Antibody (Product # G), shows increase in expression Phospho-STAT3 (Ser727) in NIH/3T3 cells treated with PDGF and A549 cells treated with EGF. Cell Treatment validation info. Phospho-STAT3 (Ser727) Antibody (44-384G) in TM Altered expression of proteins upon cell treatment demonstrates antibody specificity. Western blot of Phospho-STAT3 (Ser727) using anti-stat3 [ps727] Rabbit Polyclonal Antibody (Product # G), shows increase in expression Phospho-STAT3 (Ser727) in 3T3L1 treated with LIF. Cell Treatment validation info. Product Images For Phospho-STAT3 (Ser727) Polyclonal Antibody

3 Phospho-STAT3 (Ser727) Antibody (44-384G) in IHC (P) Immunohistochemistry analysis of STAT3 [ps727] showing staining in the cytoplasm of paraffin-embedded mouse brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (ph 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2- methanol for 15 min at room temperature, washed with ddh2o and PBS, and then probed with a STAT3 [ps727] Rabbit Polyclonal Antibody (44384G) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4 C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRPconjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

4 Phospho-STAT3 (Ser727) Antibody (44-384G) in WB Western blot analysis of STAT3 [ps727] was performed by loading 20 µg of NIH/3T3 (lane1), NIH /3T3 treated for 10 minutes with 50 ng/ml of PDGF (lane2), NIH/3T3 treated for 10 minutes with 200 ng/ml of EGF (lane3), A549 (lane4) and A549 treated for 10 minutes with 200 ng/ml of EGF (lane5) cell lysate using Novex NuPAGE 4-12 % Bis-Tris gel (NP0322BOX), XCell SureLock Electrophoresis System (EI0002), Novex Sharp Pre-Stained Protein Standard (LC5800), and iblot 2 Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature. STAT3 [ps727] was detected at ~83 kda using STAT3 [ps727] Rabbit Polyclonal Antibody (44384G) at 1: 1000 dilution in 5% skim milk at 4 C overnight on a rocking platform. Goat Anti-Rabbit IgG - HRP Secondary Antibody (G21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Novex ECL Chemiluminescent Substrate Reagent Kit (WP20005).

5 Phospho-STAT3 (Ser727) Antibody (44-384G) in WB Extracts prepared from 3T3L1 cells stimulated with LIF were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4 C, then were incubated with STAT3 [ps727] antibody (Product # G) for two hours at room temperature in a 1% BSA- TBST buffer, following prior incubation with: no peptide (1), the nonphosphopeptide corresponding to the immunogen (2), a generic phosphoserine-containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat F(ab')2 anti-rabbit IgG HRP-conjugate (Product # ALI4404) and bands were detected using the Pierce SuperSignal method. The data show that only the peptide corresponding to STAT3 [ps727] blocks the antibody signal, demonstrating the specificity of the antibody.

6 Phospho-STAT3 (Ser727) Antibody (44-384G) in ChIP ChIP- qpcr analysis of STAT3 [pser727] was performed with 10µl of the STAT3 [pser727] Rabbit polyclonal antibody (44384G) on sheared chromatin from 2 million HeLa cells treated with 100ng/ml of IFN Alpha for one hour using the MAGnify Chromatin Immunoprecipitation System ( ). Normal Rabbit IgG was used as a negative IP control. The purified DNA from each ChIP sample was analyzed by StepOnePlus Real- Time PCR System ( ) with primers for the promoter of active GLS1 gene, used as positive control target, and the cfos, used as negative control target. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

7 PubMed References For Phospho-STAT3 (Ser727) Polyclonal Antibody 1 Immunofluorescence References Species / Dilution Human / Not Cited 8 Western Blot References Species / Dilution Human / Not Cited Summary The Journal of experimental medicine (Dec 2011; 208: 2657) "Guanylate binding protein 1 is a novel effector of EGFR-driven invasion in glioblastoma." Author(s):Li M,Mukasa A,Inda MM,Zhang J,Chin L,Cavenee W,Furnari F PubMed Article URL: Summary The Journal of experimental medicine (Dec 2011; 208: 2657) "Guanylate binding protein 1 is a novel effector of EGFR-driven invasion in glioblastoma." Author(s):Li M,Mukasa A,Inda MM,Zhang J,Chin L,Cavenee W,Furnari F PubMed Article URL: G was used in western blot to show that Cdk5 modulates leptin signaling Not Applicable / 1:500 Mouse / 1:500 Journal of molecular neuroscience : MN (Sep 2009; 39: 49) "The Cdk5/p35 kinases modulate leptin-induced STAT3 signaling." Author(s):He Y,Kastin AJ,Hsuchou H,Pan W PubMed Article URL: G was used in western blot to study interactions between leptin and alpha-melanocyte stimulating hormone signaling. Journal of neurochemistry (Jul 2009; 110: 390) "Melanocortin potentiates leptin-induced STAT3 signaling via MAPK pathway." Author(s):Zhang Y,Wu X,He Y,Kastin AJ,Hsuchou H,Rosenblum CI,Pan W PubMed Article URL: G was used in western blot to investigate the role of the Wnt/beta-catenin-signaling pathway in thyrocytes Not Applicable / Not Cited Mouse / Not Cited Human / Not Cited Mouse / Not Cited Mouse / Not Cited 1 Immunohistochemistry References Species / Dilution Mouse / Not Cited The Journal of endocrinology (Apr 2007; 193: 93) "Overexpression of Wnt-1 in thyrocytes enhances cellular growth but suppresses transcription of the thyroperoxidase gene via different signaling mechanisms." Author(s):Kim WB,Lewis CJ,McCall KD,Malgor R,Kohn AD,Moon RT,Kohn LD PubMed Article URL: Molecular and cellular endocrinology (Sep 2002; 195: 109) "Protein tyrosine phosphatase 1B negatively regulates leptin signaling in a hypothalamic cell line." Author(s):Kaszubska W,Falls HD,Schaefer VG,Haasch D,Frost L,Hessler P,Kroeger PE,White DW,Jirousek MR,Trevillyan JM PubMed Article URL: The Journal of biological chemistry (Sep 2002; 277: 33509) "The serine protease plasmin triggers expression of MCP-1 and CD40 in human primary monocytes via activation of p38 MAPK and janus kinase (JAK)/STAT signaling pathways." Author(s):Burysek L,Syrovets T,Simmet T PubMed Article URL: The Journal of biological chemistry (Dec 2001; 276: 46204) "The ribosomal S6 kinases, camp-responsive element-binding, and STAT3 proteins are regulated by different leukemia inhibitory factor signaling pathways in mouse embryonic stem cells." Author(s):Boeuf H,Merienne K,Jacquot S,Duval D,Zeniou M,Hauss C,Reinhardt B,Huss-Garcia Y,Dierich A,Frank DA, Hanauer A,Kedinger C PubMed Article URL: The Journal of biological chemistry (Nov 1998; 273: 31408) "Activation of signal transducers and activators of transcription 1 and 3 by leukemia inhibitory factor, oncostatin-m, and interferon-gamma in adipocytes." Author(s):Stephens JM,Lumpkin SJ,Fishman JB PubMed Article URL: Summary PloS one (Nov 2009; 4: null) "Early-onset and robust amyloid pathology in a new homozygous mouse model of Alzheimer's disease." Author(s):Willuweit A,Velden J,Godemann R,Manook A,Jetzek F,Tintrup H,Kauselmann G,Zevnik B,Henriksen G,Drzezga A, Pohlner J,Schoor M,Kemp JA,von der Kammer H PubMed Article URL:

8 1 Immunoprecipitation References Species / Dilution Human / Not Cited Summary The Journal of biological chemistry (Sep 2002; 277: 33509) "The serine protease plasmin triggers expression of MCP-1 and CD40 in human primary monocytes via activation of p38 MAPK and janus kinase (JAK)/STAT signaling pathways." Author(s):Burysek L,Syrovets T,Simmet T PubMed Article URL: