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Stat1

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STAT1 is a laboratory equipment product manufactured by BD. It is a device used for the detection and quantification of specific biomolecules in samples. The core function of STAT1 is to perform analytical measurements, but its detailed intended use is not provided in this unbiased and factual description.

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Lab products found in correlation

β actin, by Merck Group (7 mentions) P stat5, by Cell Signaling Technology (3 mentions) Pstat1, by BD (3 mentions) Gapdh, by Santa Cruz Biotechnology (2 mentions) Tyr705, by Cell Signaling Technology (2 mentions) Tyr701, by Cell Signaling Technology (2 mentions) Horseradish peroxidase labeled secondary antibody, by Jackson ImmunoResearch (2 mentions) Stat3, by Santa Cruz Biotechnology (2 mentions) Irdye 680, by LI COR (2 mentions) Irdye 800, by LI COR (2 mentions) Bradford assay, by Bio-Rad (2 mentions) Protease inhibitor cocktail tablet, by Roche (2 mentions) Stat2, by Santa Cruz Biotechnology (2 mentions) P stat1, by Cell Signaling Technology (2 mentions) β actin, by Cell Signaling Technology (2 mentions) Phospho stat1, by BD (2 mentions) Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions) Hoechst 33342, by Thermo Fisher Scientific (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) P stat1, by Santa Cruz Biotechnology (1 mentions)

19 protocols using stat1

1

Western Blot Analysis of STAT Proteins

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Whole-cell Brij protein lysis and Western-blot analyses were performed as described [46 (link),54 (link)]. Antibodies used for the detection of the respective proteins and their dilutions were: FLAG (M2, SIGMA F-1804; mouse monoclonal; 1:500), pSTAT1 (sc-135648, Santa-Cruz Biotechnology; rabbit polyclonal; 1:1000), pSTAT2 (sc-21689-R, Santa-Cruz Biotechnology; rabbit polyclonal; 1:1000), STAT1 (C-terminus, S21120, BD Transduction Laboratories; mouse monoclonal; 1:1000), STAT2 (C-20, sc-476, Santa-Cruz Biotechnology; rabbit polyclonal; 1:1000), pSTAT5 (#9351, Cell Signaling Technology; rabbit polyclonal; 1:1000), STAT5A (L-20, sc-1081, Santa-Cruz Biotechnology; rabbit polyclonal; 1:1000), STAT5A+B (C-17, sc-835, Santa-Cruz Biotechnology; rabbit polyclonal; 1:1000), α-tubulin (DM1A, sc-32293, Santa-Cruz Biotechnology; mouse monoclonal; 1:200), Anti-Rabbit IgG-Peroxidase (SIGMA A-0545; 1:10,000), Anti-Mouse IgG-Peroxidase (SIGMA A-8924; 1:10,000). Apparent molecular weight of detected proteins was as predicted by the antibody manufacturers. Immunoblots shown are representative of at least two independent experiments. Uncropped original blots are available in the S1 File.
Michl C., Vivarelli F., Weigl J., De Nicola G.R., Canistro D., Paolini M., Iori R, & Rascle A. (2016). The Chemopreventive Phytochemical Moringin Isolated from Moringa oleifera Seeds Inhibits JAK/STAT Signaling. PLoS ONE, 11(6), e0157430.
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2

Western Blot Analysis of STAT1 Signaling

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The detailed immunoblotting protocol has been described previously [21 (link)]. Pellet of PBMCs was lysed with radioimmunoprecipitation assay (RIPA) buffer, and 7 μg of the cell lysate was loaded onto sodium dodecyl sulfate-polyacrylamide gels. The blotted membrane was blocked in 5% non-fat dry milk diluted in tris-buffered saline for 1.5 h at room temperature. Primary antibodies were diluted in the blocking buffer, and the membrane was incubated with the diluted primary antibodies for 19 h at 4°C. After washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 30 min at room temperature. After washing, the ImageQuant LAS 4000 (GE Healthcare, Hatfield, UK) was used to obtain the images. The antibodies used are as follows: STAT1 (1:1,000, rabbit, BD transduction laboratories, San Jose, CA, USA), PY-STAT1 (1:1,000, mouse, BD transduction laboratories), GAPDH (1:1,000, rabbit, Santa Cruz Biotechnology, Santa Cruz, CA, USA), HRP-conjugated rabbit immunoglobulin G (IgG) (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and HRP-conjugated mouse IgG (1:5,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Sung P.S., Lee E.B., Park D.J., Lozada A., Jang J.W., Bae S.H., Choi J.Y, & Yoon S.K. (2018). Interferon-free treatment for hepatitis C virus infection induces normalization of extrahepatic type I interferon signaling. Clinical and Molecular Hepatology, 24(3), 302-310.
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3

Interferon Signaling in Cells

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Human IFN-α (Roferon-A) was a gift from Roche. IFN-λ2 was purchased from PeproTech, Inc. The following antibodies were used for Western blotting and immunofluorescence staining: phospho-STAT1 and phospho-STAT1 Alexa Fluor 555 conjugated (Cell Signaling Technology), STAT1 (BD), β-actin (Sigma-Aldrich), and IFN-λR1 (ProSci Inc.).
Duong F.H., Trincucci G., Boldanova T., Calabrese D., Campana B., Krol I., Durand S.C., Heydmann L., Zeisel M.B., Baumert T.F, & Heim M.H. (2014). IFN-λ receptor 1 expression is induced in chronic hepatitis C and correlates with the IFN-λ3 genotype and with nonresponsiveness to IFN-α therapies. The Journal of Experimental Medicine, 211(5), 857-868.
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4

Western Blot for Phosphorylated STAT1 and STAT3

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Tissue was homogenized in lysis buffer containing 100 mM NaCl, 50 mM Tris (pH 7.5), 1 mM EDTA, 0.1% TX-100, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM vanadate, and 1x protease inhibitor cocktail tablets (Roche Diagnostics GmbH). Samples were lysed on ice at 4°C for 30 min and centrifuged for 5 min at 15,000 rpm at 4°C. Protein concentration was determined using the Bradford assay (Bio-Rad, Munich, Germany). 20 μg of protein was separated on sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred on nitrocellulose membrane. The membranes were blocked with 3% bovine serum albumin (BSA) in TRIS-buffered saline with 0.05% Tween-20 (TBST). Proteins were detected with primary antibodies specific for phospho-STAT1 (Tyr701, Cell Signaling Technology), STAT1 (Transduction Laboratories, BD Biosciences Pharmingen), phospho-STAT3 (Tyr705, Cell Signaling Technology), STAT3 (Santa Cruz Biotechnology) and β-actin (Sigma-Aldrich Chemie GmbH). Fluorescent secondary goat anti-mouse (IRDye 680) or anti-rabbit (IRDye 800) antibodies (Li-Cor Biosciences) were used with the Odyssey infrared imaging system. Alternatively, horseradish peroxidase-labeled secondary antibodies (Jackson Immunoresearch) were used with the chemiluminescence detection system from Pierce.
Hernández P.P., Mahlakoiv T., Yang I., Schwierzeck V., Nguyen N., Guendel F., Gronke K., Ryffel B., Hoelscher C., Dumoutier L., Renauld J.C., Suerbaum S., Staeheli P, & Diefenbach A. (2015). Interferon-λ and interleukin-22 cooperate for the induction of interferon-stimulated genes and control of rotavirus infection. Nature immunology, 16(7), 698-707.
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5

Western Blot for Phosphorylated STAT1 and STAT3

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Tissue was homogenized in lysis buffer containing 100 mM NaCl, 50 mM Tris (pH 7.5), 1 mM EDTA, 0.1% TX-100, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM vanadate, and 1x protease inhibitor cocktail tablets (Roche Diagnostics GmbH). Samples were lysed on ice at 4°C for 30 min and centrifuged for 5 min at 15,000 rpm at 4°C. Protein concentration was determined using the Bradford assay (Bio-Rad, Munich, Germany). 20 μg of protein was separated on sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred on nitrocellulose membrane. The membranes were blocked with 3% bovine serum albumin (BSA) in TRIS-buffered saline with 0.05% Tween-20 (TBST). Proteins were detected with primary antibodies specific for phospho-STAT1 (Tyr701, Cell Signaling Technology), STAT1 (Transduction Laboratories, BD Biosciences Pharmingen), phospho-STAT3 (Tyr705, Cell Signaling Technology), STAT3 (Santa Cruz Biotechnology) and β-actin (Sigma-Aldrich Chemie GmbH). Fluorescent secondary goat anti-mouse (IRDye 680) or anti-rabbit (IRDye 800) antibodies (Li-Cor Biosciences) were used with the Odyssey infrared imaging system. Alternatively, horseradish peroxidase-labeled secondary antibodies (Jackson Immunoresearch) were used with the chemiluminescence detection system from Pierce.
Hernández P.P., Mahlakoiv T., Yang I., Schwierzeck V., Nguyen N., Guendel F., Gronke K., Ryffel B., Hoelscher C., Dumoutier L., Renauld J.C., Suerbaum S., Staeheli P, & Diefenbach A. (2015). Interferon-λ and interleukin-22 cooperate for the induction of interferon-stimulated genes and control of rotavirus infection. Nature immunology, 16(7), 698-707.
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6

Immunoblotting of STAT Proteins

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Total cell lysates were prepared in lysis buffer (20 mM Hepes, pH 7.4, 20 mM NaCl, 10% Glycerol, and 1% Triton X-100) and protease and phosphatase inhibitor cocktails (CalBiochem). Blots were probed with antibodies that are specific for p-STAT1 (Y701, BD Biosciences), STAT1 (BD Biosciences), STAT2 (Santa Cruz), STAT3 (BD Biosciences), STAT4 (Santa Cruz), STAT5 (BD Biosciences), STAT6 (BD Biosciences), GAPDH (Santa Cruz), and β-actin (sigma).
Xiao W., Klement J.D., Lu C., Ibrahim M.L, & Liu K. (2018). IFNAR1 controls autocrine type I interferon regulation of PD-L1 expression in myeloid-derived suppressor cells. Journal of immunology (Baltimore, Md. : 1950), 201(1), 264-277.
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7

Immunoblot Analysis of STAT Proteins

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Immunoblots were prepared as described41 (link) using Abs for STAT3 (79D7, #4904), pSTAT3 (Y705, #9145), STAT5 (#9363), pSTAT5 (Y694, 9351), pSTAT1 (Y701, #9171), β-actin (#4967) (Cell Signaling Technology, Danvers, MA, USA), or STAT1 (#610185) (BD Transduction Labs, San Jose, CA, USA). Following incubation with horseradish peroxidase-conjugated secondary Ab, immune complexes were detected via SuperSignal® West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA).
Ware M.B., Zaidi M.Y., Yang J., Turgeon M.K., Krasinskas A., Mace T.A., Keenan K., Farren M.R., Ruggieri A.N., Li Y., Zhang C., Chen Z., Young G.S., Elnaggar O., Che Z., Maithel S.K., Bekaii-Saab T., El-Rayes B, & Lesinski G.B. (2020). Suppressive myeloid cells are expanded by biliary tract cancer-derived cytokines in vitro and associate with aggressive disease. British Journal of Cancer, 123(9), 1377-1386.
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8

Western Blot Analysis of Cell Lysates

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Western blot analysis of whole-cell lysates from frozen or freshly processed cell pellets was performed as described previously (Waibel et al. 2013 (link)) using primary antibodies against pJAK2Y1007/1008 (3776), JAK2 (3230), pSTAT5Y694 (9351), STAT5 (9363), pSTAT1Y701 (7649S), pS6S240/244 (2215S), S6 (2317), pERKT202/Y204 (9101), ERK (9107), c-Myc (Cell Signaling Technology, 9402), STAT1 (BD Biosciences, 610185), GFP (Invitrogen, A6455), 200 µL of β-actin (Sigma-Aldrich. A2228), Hsp90 (Enzo, ADI-SPA-830), and tubulin (Sigma-Aldrich, T5168). Immunoreactive bands were revealed using ECL reagents (Amersham ECL or ECL Prime, GE Healthcare) by film exposure (Fujifilm Super RX, Fujifilm) using an Agfa CP1000 developer (Agfa).
Kim S.K., Knight D.A., Jones L.R., Vervoort S., Ng A.P., Seymour J.F., Bradner J.E., Waibel M., Kats L, & Johnstone R.W. (2018). JAK2 is dispensable for maintenance of JAK2 mutant B-cell acute lymphoblastic leukemias. Genes & Development, 32(11-12), 849-864.
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9

Immunostaining of Transcription Factors

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Experiments were performed in 96-well plates. Cells were washed 1× with warm Dulbecco’s phosphate-buffered saline (PBS) and then fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Cells were then washed with PBS and permeabilized with 0.5% Triton X-100 in PBS at room temperature for 10 min. Cells were washed, blocked with 3% bovine serum albumin (BSA) in PBS for 30 min, and then incubated in primary antibody in antibody buffer (PBS with 0.3% Triton X-100 and 1% BSA) overnight at 4°C. The next day, cells were washed and incubated with secondary antibodies and Hoechst 33342 (2 μg/ml; Thermo Fisher Scientific #H3570) in antibody buffer for 2 hours at room temperature. After this, cells were washed with PBS and imaged in TBS-T [0.1% Tween 20 in 1× tris-buffered saline (pH 7.4)] on PerkinElmer Operetta High-Content Imaging System (25 fields per well, 20× objective, nonconfocal mode). The following antibodies were used: IRF1 (Cell Signaling Technology #8478), STAT1 (BD Biosciences #610186), and phospho-STAT1 (BD Biosciences #612133).
Sun X., Bieber J.M., Hammerlindl H., Chalkley R.J., Li K.H., Burlingame A.L., Jacobson M.P., Wu L.F, & Altschuler S.J. (2022). Modulating environmental signals to reveal mechanisms and vulnerabilities of cancer persisters. Science Advances, 8(4), eabi7711.
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10

Immunoblot Analysis of Signaling Pathways

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For immunoblot analysis, 1 × 106 neutrophils from wild-type or Ifnlr1−/− mice were stimulated as indicated in the figure legends and were lysed in 100 μl of RIPA buffer supplemented with Protease and phosphatase inhibitor cocktail (Pierce) and diisopropylfluorophosphate (Sigma). Immunoblot analysis was performed using standard techniques. Blots were probed for STAT1 phosphorylated at Tyr701 (clone 14/P-STAT1, BD), STAT2 phosphorylated at Tyr690 (Cat. No. ab53132, Abcam), STAT3 phosphorylated at Tyr705 (Cat. No. 9131, Cell Signaling Technologies), β-actin (clone AC-74, Sigma), Jak2 phosphorylated at Tyr1007 and Tyr1008 (Cat. No. sc-21870, Santa Cruz Biotechnology), Jak2 (clone C20, Santa Cruz Biotechnology), AKT (Cat. No. 9272, Cell Signaling Technology), AKT phosphorylated at Thr308 (clone 244F9, Cell Signaling Technology), AKT phosphorylated at Ser473 (clone 736E11, Cell Signaling Technology), p38 phosphorylated at Thr180 and Tyr182 (clone 36/p38, BD), STAT-1 (Cat. No. 9172, Cell Signaling Technology) and anti-puromycin (clone 12D10, Millipore).
Broggi A., Tan Y., Granucci F, & Zanoni I. (2017). IFN-λ suppresses intestinal inflammation by non-translational regulation of neutrophil function. Nature immunology, 18(10), 1084-1093.
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