Pstat1 s727

Manufactured by Cell Signaling Technology

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PSTAT1-S727 is an antibody that specifically recognizes STAT1 protein phosphorylated at serine 727. It is used in research applications to detect and quantify the phosphorylation state of STAT1 protein.

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9 protocols using pstat1 s727

Immunohistochemical Analysis of STAT1 in Ovarian Tumors

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A human ovarian tissue microarray was obtained from Alena Biotechnology Co., Ltd. (Cat# OV1005a, Xi’an, Shanxi, China). All tissues were 10% formalin-fixed and paraffin-embedded. A total of 100 ovarian tissue specimens (20 normal controls and 80 ovarian tumors) were examined by immunohistochemistry (IHC). Among 100 specimens in a slide, seven came off during the IHC staining process. In the end, 20 normal controls (3 from normal ovaries and 17 from adjacent normal ovary tissues) with median age 48.5 years (range 19–63 years) and 73 ovarian tumors (12 benign, 7 borderline, 44 malignant, 10 metastatic) with median age 49.0 years (range 17–75 years) were statistically analyzed.
After blocking with 10% normal goat serum (Fuzhou Maixin Biotech Co., Ltd., Maixin Bio, Fuzhou, Fujian, China), the sections were incubated with rabbit monoclonal antibodies against STAT1 (Cat# 9175), pSTAT1-Y701 (Cat# 9167) and pSTAT1-S727 (Cat# 8826) (Cell Signaling Technology, Inc., Danvers, MA, USA), respectively, overnight, followed by incubation with biotinylated anti-rabbit secondary antibody (Cell Signaling Technology) at room temperature for 1 h. Scoring of STAT1 immunoreactive staining was performed by two independent examiners without any prior view of patient’s clinical data and classified as described previously using staining index (SI) system [21 (link)].

Tian X., Guan W., Zhang L., Sun W., Zhou D., Lin Q., Ren W., Nadeem L, & Xu G. (2018). Physical interaction of STAT1 isoforms with TGF-β receptors leads to functional crosstalk between two signaling pathways in epithelial ovarian cancer. Journal of Experimental & Clinical Cancer Research : CR, 37, 103.

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Comprehensive Antibody Inventory for Cardiac Research

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The following primary antibodies were used for immunofluorescence and immunoblot analyses as well as coimmunoprecipitation experiments: Stat1 (Cell Signaling, 9172), p-Stat1 S727 (Cell Signaling, 9177), p-Stat1 Y701 (Cell Signaling, 9167), Smad2/3 (Cell Signaling, 5678); Smad3 and p-Smad3 (Cell Signaling [ref. 22 (link)]), SRF (Cell Signaling, 5147), Gata-4 (Cell Signaling, 36966 or Developmental Studies Hybridoma Bank [DSHB]; CRP-Gata-4-1A7, deposited to the DSHB by Protein Capture Reagents Program, produced by JHU/CDI), p-Histone H3 S10 (Cell Signaling, 3377), JAK1 (Cell Signaling, 3332), STAT3 (Cell Signaling, 9404), GAPDH (Santa Cruz Biotechnology, sc-32233), titin T11 (MilliporeSigma, T9030), sarcomeric myosin (DSHB; A4.1025), troponin-I (Cell Signaling, 4002), sarcomeric α-actinin-2 (clone EA-53, Mob 227-05, Diagnostic Biosystems), cardiac actin (Progen Biotechnik, Ac1–20.4.2), PTEN (Cell Signaling, 9188), FoxO1 (Cell Signaling, 2880), normal rabbit IgG (Santa Cruz Biotechnology, sc-2027), normal mouse IgG (Santa Cruz Biotechnology, sc-2025). Secondary fluorescence dye–linked or horseradish peroxidase–linked antibodies were obtained from Jackson Immunoresearch, DAKO, Cell Signaling or Santa Cruz Biotechnology. Fluorescently labeled phalloidin and DAPI was purchased from Molecular Probes (Life Technologies).

Lange S., Banerjee I., Carrion K., Serrano R., Habich L., Kameny R., Lengenfelder L., Dalton N., Meili R., Börgeson E., Peterson K., Ricci M., Lincoln J., Ghassemian M., Fineman J., del Álamo J.C, & Nigam V. (2019). miR-486 is modulated by stretch and increases ventricular growth. JCI Insight, 4(19), e125507.

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SARS-CoV-2 Nucleocapsid Protein Detection

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Human cell lines were grown to 70% confluence and lysed in 1X RIPA buffer (50mM Tris-HCl pH 7.4, 150mM NaCl, 1mM EDTA, 0.1% SDS, 1% NP-40, 0.25% sodium deoxycholate) supplemented with EDTA-free protease inhibitor cocktail (Roche). Proteins were separated by SDS-PAGE on 4–12% Bis-Tris gels, transferred to a nitrocellulose membranes, blocked in Intercept (PBS) blocking buffer (LI-COR) or 5% milk, and incubated with primary antibodies overnight at 4°C. Washed membranes were incubated for 45 min to 1 hour at room temperature in secondary antibody solution (LI-COR IRDye 680, 800; 1:10,000 in 5% milk or Intercept (PBS) blocking buffer), imaged on an Odyssey^® CLx scanner, and analyzed using the Image Studio Software. Antibodies were used at the following dilutions: ACE2 (R&D Systems #AF933, 1:200), β-actin (Sigma #A5316, 1:5000 or Santa Cruz Biotech #sc8432, 1:500), Vinculin (Santa Cruz #sc-73614, 1:2000), p STAT1-Y701 (Cell Signaling #9167, 1:1000), p STAT1-S727 (Cell Signaling #8826, 1:1000), STAT1 (Cell Signaling #14994, 1:1000), MX1 (Cell Signaling #37849, 1:1000), IFIT1 (Cell Signaling #14769, 1:1000), STING (Cell Signaling #13647S, 1:1000), cGAS (Cell Signaling #15102S, 1:1000), TBK1 (Cell Signaling #38066S, 1:1000), IRF3 (Cell Signaling #4302S, 1:1000), SARS-CoV-2 Nucleocapsid (Sino Biological # 40588-T62, 1;1000 or # 40143-MM05, 1:1000).

Puray-Chavez M., LaPak K.M., Jasuja R., Pan J., Xu J., Eschbach J.E., Mohammed S., Lawson D.Q., Wang Q., Brody S.L., Major M.B., Goldfarb D, & Kutluay S.B. (2024). A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models. bioRxiv.

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Protein Expression Analysis by Western Blot

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Cellular protein extracts were isolated by directly adding Laemmli sample buffer containing 5% beta mercaptoethanol to the cells. Protein concentrations were measured using Bradford protein assay. Equal amounts of protein extracts (50μg) were separated by Mini Protean precast gels TGX (Biorad). Afterwards, protein extracts were transferred to nitrocellulose membrane using a Trans-Blot® TurboTM transfer Pack (biorad) and Biorad semi-dry Transfer System. Membranes were blocked for 1 h in 5 mL 5% BSA/0.1% Tween/TBS and probed overnight with primary antibody for pSTAT1 Y701 (Cell Signaling #9167), pSTAT1 S727 (Cell Signaling #9177), Total-STAT1 (Cell Signaling #9172), IRF-1 (Cell Signaling #14028), Phospho-AKT (Cell Signaling #9271) or beta-actin (Cell Signaling #3700). membranes were washed 3 times with 5 mL 0.1% Tween/TBS and incubated with anti-rabbit IgG or anti-mouse IgG with HPR-linked antibody (Cell signaling #7074, #7075) for 1 h. Membranes were developed using 1X SignalFireTM ECL Reagent (Cell signaling) and analyzed with a compatible imager. Western-blots were quantified by measuring protein band intensity using imageJ software. Samples were normalized with beta-actin band intensity.

Marijt K.A., Sluijter M., Blijleven L., Tolmeijer S.H., Scheeren F.A., van der Burg S.H, & van Hall T. (2019). Metabolic stress in cancer cells induces immune escape through a PI3K-dependent blockade of IFNγ receptor signaling. Journal for Immunotherapy of Cancer, 7, 152.

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Protein Interaction Analysis by Co-IP and WB

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Co-immunoprecipitation, immunoprecipitation and Western blot analysis were performed as described previously [5 (link)]. Antibodies reactive with human β-actin, caspase-9, caspase-3, Bcl-2, Bcl-xL, STAT1, phospho-STAT1 serine 727 (or p-STAT1 S727), phospho-STAT1 tyrosine 701 (or p-STAT1 Y701), phospho-ERK (or p-ERK), ERK, Ubiquitin (or Ub), phospho-JAK2 (or p-JAK2), JAK2, Human influenza hemagglutinin (or HA) and Poly ADP ribose polymerase (PARP) were purchased from Cell Signaling.

Zhang Y., Chen Y., Liu Z, & Lai R. (2018). ERK is a negative feedback regulator for IFN-γ/STAT1 signaling by promoting STAT1 ubiquitination. BMC Cancer, 18, 613.

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Quantifying Immune Signaling Proteins

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Cell lysis, SDS‐PAGE, and Western Blots were performed as described previously [23]. Detection of chemiluminescence was performed using Clarity^TM Western ECL substrate (BioRad) by ChemiDocT XRS+ Molecular Imager (BioRad) and analyzed by using the Image Lab software (BioRad). Antibodies against perforin (#3693), pSTAT1^Y701 (#9167S), pSTAT1^S727 (#9177), STAT1 (#9172), pSTAT3^Y705 (#9131), STAT3 (#12640), GrzmB (#4275S), rabbit IgG (HRP linked, #7074S), and mouse IgG (HRP linked, #7076S) were purchased from Cell Signaling Technology. Antibodies against β‐actin (#47778), HSC‐70 (#7298), and STAT5 (#836) were purchased from Santa Cruz Biotechnology. Antibodies against pSTAT4^Y693 (38/p‐Stat4), STAT4 (8/Stat4), and pSTAT5^Y694 (47/Stat5(pY694)) were purchased from BD Biosciences.

Prinz D., Klein K., List J., Knab V.M., Menzl I., Leidenfrost N., Heller G., Polić B., Putz E.M., Witalisz‐Siepracka A., Sexl V, & Gotthardt D. (2020). Loss of NKG2D in murine NK cells leads to increased perforin production upon long‐term stimulation with IL‐2. European Journal of Immunology, 50(6), 880-890.

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Western Blot Analysis of SARS-CoV-2 Infection

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Human cell lines were grown to 70% confluence and lysed in RIPA (10% glycerol, 50mM Tris-HCl pH 7.4, 150mM NaCl, 2mM EDTA, 0.1% SDS, 1% NP40, 0.2% sodium deoxycholate) containing protease and phosphatase inhibitors (Thermo Scientific). Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, blocked in 5% milk, and incubated with primary antibodies overnight at 4°C. Washed membranes were incubated for 45 min at room temperature in secondary antibody solution (LI-COR IRDye 680, 800; 1:10,000 in 5% milk), imaged on an Odyssey® CLx, and analyzed using Image Studio Software. Antibodies were used at the following dilutions: ACE2 (R&D Systems #AF933, 1:200), β-actin (Sigma #A5316, 1:5000), Vinculin (Santa Cruz #sc-73614, 1:2000), AAK1 (Bethyl #A302–146A, 1:1000), AP2M1 (Abcam #ab75995, 1:1000), pAP2M1-T156 (Cell Signaling #3843, 1:1000), SARS-CoV-2-N (Sino Biological #40588-T62, 1:500), p STAT1-Y701 (Cell Signaling #9167, 1:1000), p STAT1-S727 (Cell Signaling #8826, 1:1000), STAT1 (Cell Signaling #14994, 1:1000), MX1 (Cell Signaling #37849, 1:1000), IFIT1 (Cell Signaling #14769, 1:1000), pIKKα/β-S176/180 (Cell Signaling #2697, 1:1000), IKKα (Cell Signaling #11930, 1:1000), IKKβ (Cell Signaling #8943, 1:1000), pNFKB p65-S536 (Cell Signaling #3033, 1:1000), NFKB p65 (Cell Signaling #8242, 1:1000),

Puray-Chavez M., LaPak K.M., Schrank T.P., Elliott J.L., Bhatt D.P., Agajanian M.J., Jasuja R., Lawson D.Q., Davis K., Rothlauf P.W., Jo H., Lee N., Tenneti K., Eschbach J.E., Mugisha C.S., Vuong H.R., Bailey A.L., Hayes D.N., Whelan S.P., Horani A., Brody S.L., Goldfarb D., Major M.B, & Kutluay S.B. (2021). Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell. bioRxiv.

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Western Blot Analysis of Signaling Proteins

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Antibodies for STAT3, p-STAT3-S727, p-STAT3-Y705, Mcl-1, Ran, STAT1, p-STAT1-Y701, p-STAT1-S727, STAT2, p-STAT2-Y690, STAT5, Akt-S473, BTK, p-BTK-Y223, p-ERK (T202/Y204), ERK, p-MARCKS (Ser 152/156), MARCKS, survivin, XIAP, cyclin D1, p21 and β-actin were purchased from Cell Signaling Technology Inc (Danvers, MA, USA). The anti-FLAG antibody was purchased from Sigma (St Louis, MO, USA). For western blotting, precasted Nupage electrophoresis gels were purchased from Invitrogen (Carlsbad, CA, USA) and chemiluminescence reagent was obtained from Thermo Scientific (Waltham, MA, USA). STAT3 inhibitor, Stattic; MEK inhibitor, U0126 and PKC inhibitor, Bis-I were purchased from Sigma. BTK inhibitor, ibrutinib, was purchased from MedChem Express (Monmouth Junction, NJ, USA).

Doshi U.A., Shaw J., Fox T.E., Claxton D.F., Loughran T.P, & Kester M. (2017). STAT3 mediates C6-ceramide-induced cell death in chronic lymphocytic leukemia. Signal Transduction and Targeted Therapy, 2, 17051-.

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SARS-CoV-2 Infection Signaling Pathway Analysis

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Human cell lines were grown to 70% confluence and lysed in RIPA (10% glycerol, 50mM Tris-HCl pH 7.4, 150mM NaCl, 2mM EDTA, 0.1% SDS, 1% NP40, 0.2% sodium deoxycholate) containing protease and phosphatase inhibitors (Thermo Scientific). Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, blocked in 5% milk, and incubated with primary antibodies overnight at 4°C. Washed membranes were incubated for 45 min at room temperature in secondary antibody solution (LI-COR IRDye 680, 800; 1:10,000 in 5% milk), imaged on an Odyssey® CLx, and analyzed using Image Studio Software. Antibodies were used at the following dilutions: ACE2 (R&D Systems #AF933, 1:200), β-actin (Sigma #A5316, 1:5000), Vinculin (Santa Cruz #sc-73614, 1:2000), AAK1 (Bethyl #A302-146A, 1:1000), AP2M1 (Abcam #ab75995, 1:1000), pAP2M1-T156 (Cell Signaling #3843, 1:1000), SARS-CoV-2-N (Sino Biological #40588-T62, 1:500), p STAT1-Y701 (Cell Signaling #9167, 1:1000), p STAT1-S727 (Cell Signaling #8826, 1:1000), STAT1 (Cell Signaling #14994, 1:1000), MX1 (Cell Signaling #37849, 1:1000), IFIT1 (Cell Signaling #14769, 1:1000), pIΚΚα/β-S176/180 (Cell Signaling #2697, 1:1000), IΚΚα (Cell Signaling #11930, 1:1000), IΚΚβ (Cell Signaling #8943, 1:1000), p NFΚB p65-S536 (Cell Signaling #3033, 1:1000), NFΚB p65 (Cell Signaling #8242, 1:1000),

Puray-Chavez M., LaPak K.M., Schrank T.P., Elliott J.L., Bhatt D.P., Agajanian M.J., Jasuja R., Lawson D.Q., Davis K., Rothlauf P.W., Liu Z., Jo H., Lee N., Tenneti K., Eschbach J.E., Shema Mugisha C., Cousins E.M., Cloer E.W., Vuong H.R., VanBlargan L.A., Bailey A.L., Gilchuk P., Crowe JE J.r., Diamond M.S., Hayes D.N., Whelan S.P., Horani A., Brody S.L., Goldfarb D., Major M.B, & Kutluay S.B. (2021). Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell. Cell Reports, 36(2), 109364.

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