Anti stat1

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom

Anti-STAT1 is a primary antibody that specifically recognizes the STAT1 protein. STAT1 is a transcription factor that plays a crucial role in cellular signaling pathways. This antibody can be used to detect and study the expression and activation of STAT1 in various biological samples.

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Anti-phospho-STAT1 (Y701) Anti-STAT1 (clone D1K9Y, Rabbit anti-phospho-STAT1 (pY701) and anti-STAT1 anti-sera Anti-phosphorylated STAT1 Rabbit anti-Phospho-Stat1 (Ser727) Rabbit anti-STAT1 Ab Anti-pY701-STAT1 Rabbit anti-phospho STAT1 (Tyr 701) monoclonal antibody Rabbit anti-phosphorylated STAT1 Anti-total STAT1

160 protocols using anti stat1

Immunoprecipitation and Western Blotting of STAT1 Acetylation

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Mouse lung tissue samples were lysed in RIPA buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol) with protease/phosphatase inhibitor cocktail (5872S, Cell Signaling Technology, 1:1000), 0.3 mM DTT, 5 mM nicotinamide, and 1 mM sodium butyrate. Total 500 μg of protein were used for immunoprecipitation with indicated dilution of each antibody (anti-STAT1, 14994, Cell Signaling Technology, 1:100).
Western blotting was performed according to standard protocols. The antibodies and dilutions were anti-β-actin (sc-5286, Santa Cruz, 1:10,000), anti-STAT1 (14994, Cell Signaling Technology, 1:1000), anti-acetyl-lysine (PTM-105, PTM Bio, 1:1000), and anti-succinyl-lysine (PTM-401, PTM Bio, 1:1000).

Zhang T., Wang Y., Sun Y., Song M., Pang J., Wang M., Zhang Z., Yang P., Chen Y., Qi X., Zhou H., Han Z., Xing Y., Liu Y., Li B., Liu J., Yang J, & Wang J. (2024). Proteome, Lysine Acetylome, and Succinylome Identify Posttranslational Modification of STAT1 as a Novel Drug Target in Silicosis. Molecular & Cellular Proteomics : MCP, 23(6), 100770.

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Western Blot Analysis of Signaling Proteins

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The whole-cell lysate of BMMCs was subjected to SDS-PAGE, and the separated proteins were transferred to a membrane. The blots were then incubated with the target antibody, followed by the appropriate HRP-conjugated secondary antibody, and visualized by the LAS-3000 detection system (Fuji Photo Film, Japan). The following antibodies were used for immunoblot analyses: anti-Stat1 (Cell Signaling Technology; hereafter CST, #9172), anti-p-Stat1 (CST #7649), anti-Stat3 (CST #12640), anti-p-Stat3 (CST #9145), anti-PLCγ2 (Santa Cruz Biotechnology; hereafter SCB, sc-407), anti-p-PLCγ2 (CST #3871), anti-c-kit (SCB, sc-1494), anti-p-c-kit (CST #3319), anti-Syk (SCB, sc-929), anti-p-Syk (CST #2710), anti-Src (CST #2109), anti-p-Src family (CST #6943), anti-Phosphotyrosine (4G10)-HRP (MM 16–316), anti-FcεRI β chain (MM MABF38), anti-FcεRI γ chain (MM 06–727), anti-ERK1/2 (CST #4695), anti-p-ERK1/2 (CST #4370), anti-p38(CST #9212), anti-p-p38 (CST #9211), anti-β-Actin (CST #4970), and anti-α-Tubulin (CST #2125). To quantify the band intensities, Image Gauge (Fuji Photo Film, Tokyo, Japan) was used.

Kobayashi T., Shimabukuro-Demoto S., Tsutsui H, & Toyama-Sorimachi N. (2019). Type I interferon limits mast cell–mediated anaphylaxis by controlling secretory granule homeostasis. PLoS Biology, 17(11), e3000530.

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DUSP1 Silencing Impact on STAT1 Translocation

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To assess the effect of DUSP1 silencing on translocalization of STAT1, cells expressing LV-cont or LV-shDUSP1 were seeded at a density of 2 × 10⁵ cells per plate. After 1 day of seeding, cells were fixed with 4% paraformaldehyde for 30 min, washed three times with PBS for 5 min, and incubated at 4°C overnight with a 1:50 dilution of anti-STAT1 (Cell Signaling Technology, Beverly, MA) in blocking solution (1% bovine serum albumin). Cells were washed three times with PBS, then incubated with Alexa 594-conjugated anti-rabbit IgG (Molecular Probes, Eugene, OR) for 1 h at room temperature. Nuclei were detected by staining with 1 μg/mL 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 5 min. After three washes with PBS, preparations were mounted with Kaiser’s glycerin gelatin (Merck, Darmstadt, Germany) and examined using an Axiovert fluorescence microscope (Carl Zeiss, Göttingen, Germany).

Choi J.E., Kwon J.H., Kim J.H., Hur W., Sung P.S., Choi S.W, & Yoon S.K. (2015). Suppression of Dual Specificity Phosphatase I Expression Inhibits Hepatitis C Virus Replication. PLoS ONE, 10(3), e0119172.

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Immunoblotting Analysis of Intestinal Signaling

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Mouse intestinal mucosa, including most proximal and distal regions, was collected by scraping as previously described [27] (link). Mouse epithelial cells were broken in a lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA pH 8.0, 0.2 mM sodium orthovanadate, and protease inhibitor cocktail) (Roche, Nutley, NJ). Immunoblotting was performed with primary antibodies: anti–phospho-Stat1, anti–phospho-Stat3, anti–phospho-Jak2, anti-Stat1, anti-Stat3, and anti-Jak2 (Cell Signal, Beverly, MA), or anti–beta-actin (Sigma-Aldrich, Milwaukee, WI) antibodies and secondary antibodies visualized by ECL [42] (link).

Lu R., Wu S., Zhang Y.G., Xia Y., Zhou Z., Kato I., Dong H., Bissonnette M, & Sun J. (2016). Salmonella Protein AvrA Activates the STAT3 Signaling Pathway in Colon Cancer. Neoplasia (New York, N.Y.), 18(5), 307-316.

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Isolation and Culture of Hepatic and Immune Cell Populations

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Human Huh7.5.1 cells were kindly provided by Dr. F. Chisari (The Scripps Research Institute, La Jolla, CA; MTA number 636) [18 (link)]. PHH were provided by Stephen Strom of University of Pittsburgh through the Liver Tissue Procurement and Distribution System (N01-DK-7-0004/HHSN26700700004C). PHH were isolated from liver resections according to standard perfusion protocols. After seven days of culture, PHH remained attached and maintained a healthy and highly differentiated phenotype. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy blood donors (EFS-Alsace, Strasbourg, France) by Ficoll density gradient centrifugation. CD4+ T cell positive selection was performed by magnetic sorting using CD4+ microbeads (Miltenyi Biotech). Human recombinant IFN-α and IFN-γ were obtained from Roche and Intermune, respectively. Rabbit polyclonal anti-IDO1 (Abcam), anti-β actin (Abcam), anti-IRF1 (Santa Cruz Biotechnology), anti-STAT1 and anti-STAT1 (Tyr701) antibody (Cell Signaling) were used for Western blot analysis. PE-conjugated mouse anti-human IFN-γR, anti-HCV core (clone C7-50, Thermo Scientific) and anti-IRF1 antibodies were used for flow cytometry analysis. The IDO inhibitor 1-DL-MT, a racemic mixture of 1-methyl-tryptophan comprising both isomers (D+L), was purchased from Sigma-Aldrich.

Lepiller Q., Soulier E., Li Q., Lambotin M., Barths J., Fuchs D., Stoll-Keller F., Liang T.J, & Barth H. (2015). Antiviral and Immunoregulatory Effects of Indoleamine-2,3-Dioxygenase in Hepatitis C Virus Infection. Journal of Innate Immunity, 7(5), 530-544.

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Western Blot Analysis of JAK2 Signaling

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Leukemic cells were lysed in lysis buffer supplemented with freshly added protease and phosphatase inhibitors. 25μg (BCA method; Thermo Scientific) lysate was loaded on 10% mini protean precast gels (BioRad, Veenendaal, Netherlands), and transferred to a nitrocellulose membrane (Biorad). Primary antibody incubation was performed according to manufacturer's protocol. Anti-JAK2 (#3230), anti-phospho-JAK2-Tyr1007 (#4406), anti-phospho-STAT5-Tyr694 (#9351), anti-phospho-STAT1-Tyr701 (#9167), anti-Stat1 (#9175), anti-phospho-MEK1/2-Ser217/221 (#9154), anti-MEK1/2 (#4694), anti-phospho-Erk1/2-T202/204 (#4370), anti-Erk1/2 (#91078), and anti-αTubulin (#2144) were obtained from Cell Signaling Technology (Danvers, Massachusetts, USA). Anti-β-actin (ab6276) was obtained from Abcam (Cambridge, UK), and anti-STAT5 (sc-835) from Santa Cruz (Heidelberg, Germany). Blots were stained with secondary antibodies (IRDye 680RD- or 800CW-labelled anti-rabbit and IRDye 680RD- or 800CW-labelled anti-mouse; Li-Cor Biosciences, Leusden, Netherlands) and scanned using the Odyssey infrared imaging system (Li-Cor Biosciences). To reprobe membranes, they were stripped in NewBlot Nitrocellulose stripping buffer (Li-Cor Biosciences) according to manufacturer's protocol. BCR-JAK2, PAX5-JAK2 and TERF2-JAK2 proteins were separated from wildtype JAK2 based on size (~94, 57, 95 and 125 kDa, respectively).

Steeghs E.M., Jerchel I.S., de Goffau-Nobel W., Hoogkamer A.Q., Boer J.M., Boeree A., van de Ven C., Koudijs M.J., Besselink N.J., de Groot-Kruseman H.A., Zwaan C.M., Horstmann M.A., Pieters R, & den Boer M.L. (2017). JAK2 aberrations in childhood B-cell precursor acute lymphoblastic leukemia. Oncotarget, 8(52), 89923-89938.

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Analyzing IL-33 and NF-κB Signaling

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Cells were collected after stimulation and rinsed with ice-cold PBS/phosphatase inhibitors. Proteins in the total fraction were extracted in a reduced lysis buffer [60 mM Tris-HCL (pH 6.8), 10%glycerol, and protease inhibitor cocktail]. Nuclear extracts were collected according to the instruction of the nuclear extract kit (Active Motif, Carlsbad, CA). Equal quantities of protein were separated by electrophoresis on 10% SDS-PAGE gels and subjected to western blotting with anti-IL-33 (R&D Systems) 1:1000, anti-STAT1 (Cell Signaling, Beverly, MA) 1:1000, Phospho-NF-κB p65 antibody (Cell Signaling) 1:1000 or anti-β-actin (Cell Signaling) 1:1000 overnight at 4°C. After the incubation with the appropriate secondary horseradish peroxidase-conjugated IgG antibody (R&D Systems) for 2 h at RT, the protein bands on the membrane were detected with ECL-Plus Western Blot Detection system (GE Healthcare UK LTD) according to the manufacturer’s instructions. All experiments were replicated at least three times. The results of typical experiments are shown. The western blot bands were analyzed using ImageJ software (Bio-Arts, Co. Ltd, Fukuoka, Japan).

Shan J., Oshima T., Wu L., Fukui H., Watari J, & Miwa H. (2016). Interferon γ-Induced Nuclear Interleukin-33 Potentiates the Release of Esophageal Epithelial Derived Cytokines. PLoS ONE, 11(3), e0151701.

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Western Blot Analysis of Apoptosis and Signaling

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Cell pellets were lysed in RIPA buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1% NP-40, 1 mM DTT, 1 mM NaF, 1 mM sodium vanadate, 1 mM PMSF (Sigma), and 1% protease inhibitors cocktail (Merck). Protein extracts were quantitated and loaded on 8% to 12% sodium dodecyl sulfate polyacrylamide gel, electrophoresed, and transferred onto a PVDF membrane (Millipore). The membrane was incubated with primary antibody, washed, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Pierce). Detection was performed by using a chemiluminescent Western detection kit (Cell Signaling Technology). The antibodies used were Anti-caspase-3, Anti-caspase-9, Anti-PARP, Anti-pSTAT3 (Y705), Anti-Jak2, Anti-pJak2 (Y1007/Y1008), Anti-pSTAT3 (S727), Anti-STAT1, Anti-pSTAT1 (Y701), Anti-STAT5, and Anti-pSTAT5 (Y694) (Cell Signaling Technology), Anti-Lamin B, Anti-Cyclin D1 (Santa Cruz Biotechnology), Anti-Mcl-1, Anti-STAT3 (Proteintech), and Anti-GAPDH (Abmart) antibodies.

Feng T., Cao W., Shen W., Zhang L., Gu X., Guo Y., Tsai H.I., Liu X., Li J., Zhang J., Li S., Wu F, & Liu Y. (2016). Arctigenin inhibits STAT3 and exhibits anticancer potential in human triple-negative breast cancer therapy. Oncotarget, 8(1), 329-344.

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Gut Microbiome Modulation and Immune Response

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VSL#3 packets with 450 billion bacteria per sachet (gift from Professor Claudio De Simone), human recombinant IFN-γ (Roche, Mannheim, Germany), cycloheximide (Sigma-Aldrich, St. Louis, MO, USA), monoclonal mouse anti-TCPTP antibody CF-4, which detects the 45-kilodalton and the 48-kilodalton isoforms (Calbiochem, San Diego, CA), mouse anti-TCPTP (Ab-1) antibody (EMD Millipore, Billerica, MA), anti-phospho-STAT1 (Tyr⁷⁰¹), anti-STAT1, (Cell Signaling Technologies, Danvers, MA), Claudin-2, Occludin and ZO-1 (Invitrogen, Waltham, Massachusetts, USA) and monoclonal mouse anti-β-Actin (Sigma) were obtained from the sources noted. Millicell culture plate inserts were purchased from Millipore Corporation (Millipore, Bedford, MA). McCoy’s 5A and DMEM media were purchased form Corning Inc, (Corning, NY). All other reagents were of analytical grade and acquired commercially.

Krishnan M., Penrose H.M., Shah N.N., Marchelletta R.R, & McCole D.F. (2016). VSL#3 Probiotic Stimulates T-Cell Protein Tyrosine Phosphatase-Mediated Recovery of IFN-γ Induced Intestinal Epithelial Barrier Defects. Inflammatory bowel diseases, 22(12), 2811-2823.

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Western Blotting of Transcription Factors

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Approximately 30 mg of mouse liver was homogenized and centrifuged at 16,500 g for 20 minutes at 4°C. After adding sodium dodecyl sulfate (SDS) sample buffer, the supernatants were boiled for 10 minutes at 95°C. Protein concentrations in the samples were determined by the BCA assay (Thermo Fisher Scientific Inc.). Then, appropriate amounts of protein were loaded onto 8–12% SDS–polyacrylamide gels and electrotransferred onto a polyvinylidene fluoride membrane. The membrane was blocked with 5% skimmed milk or 1% bovine serum albumin for 1 hour at room temperature and then incubated overnight at 4°C with the primary antibodies anti-STAT1, anti-phospho-STAT1, anti-IRF3, anti-phospho-IRF3 (diluted 1:1000, Cell Signaling Technology), anti-STAT2, anti-phospho-STAT2 (diluted 1:1000, Abcam, Cambridge, UK) or β-actin (diluted 1:10,000, Sigma-Aldrich, St. Louis, MO, USA). After the membrane was washed three times for 5 minutes in TBST, it was incubated with the appropriate HRP-conjugated secondary antibody (diluted 1:5000 in TBST) for 1 hour at room temperature. Blotted protein bands were visualized by enhanced chemiluminescence (Thermo Fisher Scientific Inc.) and exposed to X-ray film.

Yokoyama Y., Miyagi T., Hikita H., Yoshioka T., Mukai K., Nawa T., Sakamori R., Ohkawa K., Hiramatsu N., Takahashi T., Suemizu H., Ryo A., Tatsumi T, & Takehara T. (2015). The Hepatitis B Virus Genotype Affects the Persistence of Viral Replication in Immunodeficient NOG Mice. PLoS ONE, 10(12), e0144775.

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