Stat1 antibody

Manufactured by Cell Signaling Technology

Sourced in United States

The STAT1 antibody is a laboratory tool used to detect and study the STAT1 protein, which is a key transcription factor involved in cellular signaling pathways. This antibody can be used in various applications, such as Western blotting, immunoprecipitation, and immunohistochemistry, to investigate the expression, localization, and interactions of STAT1 in different cell types or tissues.

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Lab products found in correlation

Nf κb p65 antibody, by Abcam (2 mentions) Pierce agarose chip kit, by Thermo Fisher Scientific (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions) Hoechst 33342, by Thermo Fisher Scientific (2 mentions) Human pd l1 antibody, by Cell Signaling Technology (1 mentions) Pbrm1 antibody, by Cell Signaling Technology (1 mentions) Jak1 antibody, by Cell Signaling Technology (1 mentions) Jak2 antibody, by Cell Signaling Technology (1 mentions) Phospho jak2 antibody, by Cell Signaling Technology (1 mentions) β actin antibody a1978, by Merck Group (1 mentions) Phospho stat1 antibody, by Cell Signaling Technology (1 mentions) Human ifnγ 285 if 100, by R&D Systems (1 mentions) Novaseq 6000, by Illumina (1 mentions) Fitc mouse anti rabbit igg, by Boster Bio (1 mentions) Magna chip g kit, by Merck Group (1 mentions) Abs130136, by Absin (1 mentions) Anti phospho stat1 antibody, by Cell Signaling Technology (1 mentions) A5441, by Merck Group (1 mentions) Ab1793, by Abcam (1 mentions) Irdye 800 labeled igg, by LI COR (1 mentions)

Close spelling variants of this product

STAT1 (305 citations) Anti-STAT1 antibody (13 citations) Anti-phospho-STAT1 antibody (6 citations) Anti-STAT1 rabbit antibody (4 citations) Phospho-STAT1 antibody (2 citations) Rabbit anti-phospho-STAT1 (Tyr701) antibody (2 citations) Anti-P-STAT1 antibody (2 citations) Rabbit anti-phospho STAT1 (Tyr 701) monoclonal antibody (2 citations) STAT1 Antibody #9172 (2 citations) Phospho-STAT1 (Tyr 701) Alexa Fluor 488 conjugated rabbit antibody (2 citations)

14 protocols using stat1 antibody

Comprehensive Immune Marker Antibody Panel

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PBRM1 antibody (A301-591A) was from Bethyl Laboratories. Phospho-STAT1 antibody (clone ST1P-11A5; Tyr701; 33-3400), human CD3 antibody (clone F7.2.38; MA5-12577), human CD45RO antibody (clone UCHL1; MA5-11532), human CD4 antibody (clone 4B12; MS1528S0), and human CD8 antibody (clone C8/144B; MS457S0) were from ThermoFisher Scientific. Mouse CD3 antibody (D4V8L; 99940), mouse CD8 antibody(D4W22; 98941), mouse CD4 antibody (D7D2Z; 25229), human PD-L1 antibody (E1L3N; 13684), mouse PD-L1 antibody (D5V3B; 64988), mouse PD-1 antibody (D7D5W; 84651), PBRM1 antibody (D3F7O, 91894), JAK1 antibody (6G4, 3344), JAK2 antibody (D2E12, 3230), Phospho-JAK1 antibody (D7N4Z, Tyr1034/1035, 74129), Phospho-JAK2 antibody (C80C3, Tyr1007/1008, 3776), STAT1 antibody (D1K9Y, 14994), Phospho-STAT1 antibody (58D6, Tyr701, 9167), Phospho-STAT1 antibody (D3B7, Ser727, 8826), IRF1 antibody (D5E4, 8478), and BRG1 antibody (E9O6E; 52251) were from Cell Signaling Technology. IFNGR1 antibody (112802) was from BioLegend. IFNGR2 antibody (Cat No. GTX 64548) was from GeneTex. β-actin antibody (A1978) was from Sigma. Human IFNγ (285-IF-100) and mouse IFNγ (8234-MB-010) were from R&D Systems.

Liu X.D., Kong W., Peterson C.B., McGrail D.J., Hoang A., Zhang X., Lam T., Pilie P.G., Zhu H., Beckermann K.E., Haake S.M., Isgandrova S., Martinez-Moczygemba M., Sahni N., Tannir N.M., Lin S.Y., Rathmell W.K, & Jonasch E. (2020). PBRM1 loss defines a nonimmunogenic tumor phenotype associated with checkpoint inhibitor resistance in renal carcinoma. Nature Communications, 11, 2135.

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Stat1 ChIP-Seq in Stem Cells

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For ChIP, 4 × 10⁶ cells (differentiated and undifferentiated SCs) were treated with fresh 1% formaldehyde (#J60401, ThermoFisher) for cell cross-linking, which was terminated with 125 mM glycine. Cells were harvested and enzymatically digested to disrupt chromatin, breaking genomic DNA to 100–500 bp. Afterwards, the chromatin fragments (~ 300 µg) were incubated with Stat1 antibody (5 μg, Cell Signaling, #14994) at 4 °C overnight, followed by the addition of ChIP Grade proteinA/G beads (20 μl, #26156, ThermoFisher) for a further 4–6 h at 4 °C. The beads were then melted with proteinase K, followed by extraction using a DNA extraction kit for DNA library construction and sequencing analysis. The prepared ChIP-Seq libraries were detected using an Illumina NovaSeq 6000, and ChIP-Seq data were read and aligned to the rat genome (Rn6) using Bowtie2 v2.3.5.1. Based on the ENCODE (v90) overlap rule, the MACS version 1.4.2 (http://liulab.dfci.harvard.edu/MACS) was used to perform peak calling with a p-value cutoff of 10–9, and used the software IGV 2.9.4 to show the peaks. Furthermore, the motifs likely to be targeted by Stat1 were predicted using the HOMER v4.11 program (http://homer.salk.edu/homer). The data have been submitted to the GEO repository (GEO Series accession number: GSE211337).

Xu J., Zhang B., Cai J., Peng Q., Hu J., Askar P., Shangguan J., Su W., Zhu C., Sun H., Zhou S., Chen G., Yang X, & Gu Y. (2023). The transcription factor Stat-1 is essential for Schwann cell differentiation, myelination and myelin sheath regeneration. Molecular Medicine, 29, 79.

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Regulation of STAT1 and IRF1 by miR-103

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RAW264.7 cells were plated in 24-well culture plates at a concentration of 5×10⁴ cells/well and transfected with miR-103 mimics or NC. After 24 h, the cells were stimulated by IFNγ and LPS for 24h. Thereafter, the treated cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes and then incubated in 10% normal mice serum for 30 minutes. The cells were then incubated with STAT1 antibody (Cell Signaling Technology, Danvers, MA, USA) or IRF1 (8478, Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. The secondary antibodies FITC Mouse Anti-Rabbit IgG (BOSTER, Wuhan, China) was used for 1 hour. Sections were counterstained with DAPI and analyzed with a fluorescence microscope (IX73, Olympus, Japan).

Zhu X., Liu H., Zhang Z., Wei R., Zhou X., Wang Z., Zhao L., Guo Q., Zhang Y., Chu C., Wang L, & Li X. (2020). MiR-103 protects from recurrent spontaneous abortion via inhibiting STAT1 mediated M1 macrophage polarization. International Journal of Biological Sciences, 16(12), 2248-2264.

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ChIP-qPCR Analysis of Stat1 Binding

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Pooled differentiating Schwann cells were fixed with fresh 1% formaldehyde (J60401,ThermoFisher) for 20 min, and enzymatically lysed to break genomic DNA to 200 and 1000 bp. Sheared chromatins were incubated with 5 µg of Stat1 antibody (Cell Signaling, #14994) at 4 °C. Magna ChIP G Kit (Merck Millipore) was used to perform ChIP experiments. The DNAs obtained by Stat1 ChIP were used as the templates for quantitative real-time PCR, and the IgG group served as a control. The relative fold enrichment was calculated using the 2^−ΔCT method to determine the specific region bound by the Stat1 protein. Primers used to study gene promoter regions such as Ano1, Nts, C1qb and Rab11fip1 are provided in Additional file 5: Table S4.3.

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Chromatin Immunoprecipitation of Stat1 and NF-κB

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Chromatin from MSCs was crosslinked by formaldehyde treatment and immunoprecipitated using a Pierce Agarose ChIP kit (Thermo Fisher Scientific, Rockford, IL, USA). Purified chromatin (10 μg) was normalized to input and immunoprecipitated using 6 μg of NF-κB p65 antibody (Abcam, Cambridge, MA, USA), Stat1 antibody at 1:50 (Cell Signaling Technology, Danvers, MA, USA) or 1 μl of rabbit IgG control. Immunoprecipitated DNA was subject to quantitative PCR (qPCR) to determine enrichment of Stat1 homodimers and NF-κB binding to respective promoters and results were normalized to control group. Primers used for qPCR were as follows: binding of Stat1 homodimers to mouse iNOS promoter, forward 5′-GGCACCATCTAACCTCAC-3′ and reverse 5′-CAGCACGTAGTCACTTCA-3′; NF-κB binding to mouse iNOS promoter, forward 5′-TGAGGATACACCACAGAGT-3′ and reverse 5′-GTGCAAGTTAGCTCATTCAT-3′; binding of Stat1 homodimers to mouse Ccl5 promoter, forward 5′-TATAGGGAGCCAGGGTAGCA-3′ and reverse 5′-GCAACAAGTGTTTGGTGTCTTT-3′; NF-κB binding to mouse Ccl5 promoter, forward 5′-AGCCAGGGTAGCAGAGGAA-3′ and reverse 5′-ATGACAGCAACAAGTGTTTGGT-3′.

Shou P., Chen Q., Jiang J., Xu C., Zhang J., Zheng C., Jiang M., Velletri T., Cao W., Huang Y., Yang Q., Han X., Zhang L., Wei L., Rabson A.B., Chin Y.E., Wang Y, & Shi Y. (2016). Type I interferons exert anti-tumor effect via reversing immunosuppression mediated by mesenchymal stromal cells. Oncogene, 35(46), 5953-5962.

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Western Blot Analysis of Inflammatory Markers

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Western blot tests were performed as previously described [10 (link)]. The primary antibodies included anti-β-actin antibody (1:10000, a-5441, Sigma), anti-iNOS (1:500, abs130136, Absin), anti-TNF-α (1:500, ab1793, Abcam), anti-arginase-1 antibody (1:1000, sc-21050, Abcam), anti-STAT1 antibody (1:1000, #9172, Cell Signaling), and anti-phospho-STAT1 antibody (1:1000, #9167, Cell Signaling). The blots were then developed by horseradish peroxidase-conjugated secondary antibodies. Finally, the blots were enhanced by chemiluminescence detection before photography. The relative density of the comparable results was measured by ImageJ 1.37v software (Wayne Rasband).

He D., Liu F., Cui S., Jiang N., Yu H., Zhou Y., Liu Y, & Kou X. (2020). Mechanical load-induced H2S production by periodontal ligament stem cells activates M1 macrophages to promote bone remodeling and tooth movement via STAT1. Stem Cell Research & Therapy, 11, 112.

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Optimizing SARS-CoV-2 Gene Expression

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The 23 genes of SARS-CoV-2 (IPBCAMS-WH-01/2019 strain, no. EPI_ISL_402123) were optimized by Gene Designer 1.0 and cloned to vector pCMV6-entry expression vector with the FLAG-tag or HA-tag at C-terminus. Plasmids Flag-RIG-I, Flag-RIG-IN, Flag-MDA5, HA-MAVS, pGL3-IFN-β–Luc, IRF3-5D-Flag, and pRL-TK have been described elsewhere⁴². The mutated variants of the SARS-CoV-2 ORF6-tagged Flag were constructed by using a Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). All variants were confirmed by subsequent sequencing.
The antibodies used in this research were: Flag antibody from Sigma-Aldrich (1:4000, Cat# F3165); β-actin antibody from Sigma–Aldrich (1:4000, Cat# A5441); HA antibody from Sigma–Aldrich (1:10,000, Cat# H9658); STAT1 antibody from Cell Signaling technology (1:1000, Cat# 9172); P-STAT1 antibody from Thermo Fisher (1:1000, Cat# 700349); Sev antibody from MBL (1:2000, PD029C1). Dual-Luciferase® Reporter Assay System was purchased from Progema (Madison, WI). IRDye 800-labeled IgG and IRDye 680-labeled IgG secondary antibodies were purchased from Li-Cor Biosciences (Lincoln, NE).

Lei X., Dong X., Ma R., Wang W., Xiao X., Tian Z., Wang C., Wang Y., Li L., Ren L., Guo F., Zhao Z., Zhou Z., Xiang Z, & Wang J. (2020). Activation and evasion of type I interferon responses by SARS-CoV-2. Nature Communications, 11, 3810.

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Immunofluorescence Staining of Cellular Proteins

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Cells were washed with PBS buffer and fixed with 4% formalin. Then cells were permeabilized with 0.5% Triton X-100. After cells were washed with PBS, they were blocked and stained with primary antibodies, followed by staining with an Alexa Fluor 488 secondary antibody⁴⁵. Nuclei were stained with DAPI (Sigma). The antibodies used in this research were: IRF3 antibody from Cell Signaling technology (1:200, Cat# 11904); STAT1 antibody from Cell Signaling technology (1:400, Cat# 14994); P-STAT1 antibody from Cell Signaling technology (1:400, Cat# 9167); Calnexin antibody from Cell Signaling technology (1:50, Cat# 2679); GolgiB1 antibody from Sigma-Aldrich (1:500, Cat# HPA011008). Fluorescence images were obtained and analyzed using a laser scanning confocal microscope (Leica TCS SP5).

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ChIP-qPCR Analysis of Stat1 and NF-κB Binding

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Chromatin from MSCs was crosslinked by formaldehyde treatment and immunoprecipitated using a Pierce Agarose ChIP kit (Thermo Fisher Scientific, Rockford, IL, USA). Purified chromatin (10 μg) was normalized to input and immunoprecipitated using 6 μg of NF-κB p65 antibody (Abcam), Stat1 antibody at 1:50 (Cell Signaling Technology) or 1 μl of rabbit IgG control. Immunoprecipitated DNA was subjected to quantitative PCR to determine the enrichment of Stat1 homodimers and NF-κB binding to respective promoters, and results were normalized to control group. Primers used for quantitative PCR were as follows: binding of Stat1 homodimers to mouse iNOS promoter, forward 5′-GGCACCATCTAACCTCAC-3′ and reverse 5′-CAGCACGTAGTCACTTCA-3′ NF-κB binding to mouse iNOS promoter, forward 5′- TGAGGATACACCACAGAGT-3′ and reverse 5′-GTGCAAGTTAGCTCATTCAT-3′ binding of Stat1 homodimers to mouse Ccl5 promoter, forward 5′-TATAGGGAGCCAGGGTAGCA-3′ and reverse 5′-GCAACAAGTGTTTGGTGTCTTT-3′ NF-κB binding to mouse Ccl5 promoter, forward 5′-AGCCAGGGTAGCAGAGGAA-3′ and reverse 5′-ATGACAGCAACAAGTGTTTGGT-3′.

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STAT1 Activation Visualization Protocol

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Cells were starved overnight and treated with 100 U/ml IFNγ for 3 h. Cells were fixed with 4% paraformaldehyde for 10 min, permeabilized for 5 min with 0.3% Triton-X100, blocked with 2% BSA for 1 h. Cells were incubated with STAT1 antibody (14994, Cell Signaling Technology) overnight after which they were incubated for 1 h at RT to secondary anti-rabbit AlexaFluor 488 (Jackson ImmunoResearch), followed by incubation with Hoechst 33342 (Thermo Fisher Scientific) for 30 min. Confocal images were acquired with ZEN software on a Zeiss LSM 710 Confocal system (Carl Zeiss Inc.).

Mineo M., Lyons S.M., Zdioruk M., von Spreckelsen N., Ferrer-Luna R., Ito H., Alayo Q.A., Kharel P., Larsen A.G., Fan W.Y., Auduong S., Grauwet K., Passaro C., Khalsa J.K., Shah K., Reardon D.A., Ligon K.L., Beroukhim R., Nakashima H., Ivanov P., Anderson P.J., Lawler S.E, & Chiocca E.A. (2020). Tumor interferon signaling is regulated by a lncRNA INCR1 transcribed from the PD-L1 locus. Molecular cell, 78(6), 1207-1223.e8.

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