HeLa cells were grown on glass bottom cell culture dish (NEST) and transfected with Mpro-mCherry construct, and after 24 h of expressing, cells were treated with 1000 U/ml IFNα for 1 h, then cells were fixed in 1 ml of 4% formaldehyde for 30 min and permeabilized in 0.5% TritonX-100 for 10 min. After washing with PBS, samples were blocked with 10% goat serum for 1 h and incubated with the anti-STAT1(Cell Signaling, 14994), anti-STAT2 (Proteintech, 16674-1-AP), or anti-IRF9 (Santa Cruz, sc-365893) antibody overnight at 4 °C. Goat anti-Rabbit IgG (Alexa Fluor 488, ab150081) or Goat Anti-Mouse IgG (Alexa Fluor 488, ab150113) was used as the secondary antibody at 1:1000 dilution. DAPI (Beyotime) was used as nuclear counterstain. Images were captured by using Nikon A1R+ Inverted Microscope with a 60 × 1.4 NA oil-immersion objective.
Anti irf9
Manufactured by Santa Cruz Biotechnology
Sourced in Japan
Anti-IRF9 is a laboratory reagent used in research applications. It is an antibody that specifically targets the IRF9 protein, which is involved in the regulation of gene expression and cellular signaling pathways. The core function of this product is to enable researchers to detect and study the IRF9 protein in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti irf3, by Santa Cruz Biotechnology (2 mentions)
A1r inverted microscope, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Glass bottom cell culture dish, by NEST Biotechnology (1 mentions)
Normal goat igg, by Abcam (1 mentions)
Normal rabbit igg, by Cell Signaling Technology (1 mentions)
Anti irf1, by Santa Cruz Biotechnology (1 mentions)
Anti irf7, by Santa Cruz Biotechnology (1 mentions)
Anti irf8, by Santa Cruz Biotechnology (1 mentions)
Anti irf5, by Abcam (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Simplechip enzymatic chromatin ip kit, by Cell Signaling Technology (1 mentions)
Ab4086, by Abcam (1 mentions)
Anti pol 2, by Santa Cruz Biotechnology (1 mentions)
Anti phospho tyr701 stat 1, by Cell Signaling Technology (1 mentions)
Anti acetyl histone h3, by Merck Group (1 mentions)
Anti histone h1, by Abcam (1 mentions)
Anti histone h3, by Abcam (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti stat1, by Cell Signaling Technology (1 mentions)
5 protocols using anti irf9
1
Visualizing IFNα-induced STAT1/2/IRF9 in HeLa cells
2
ChIP Assay Protocol for IRF Transcription Factors
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ChIP assays were performed with SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology Japan, Tokyo, Japan) with anti-IRF1 (Santa Cruz Biotechnology), anti-IRF3(Santa Cruz Biotechnology), anti-IRF5 (Abcam), anti-IRF7 (Santa Cruz Biotechnology), anti-IRF8 (Santa Cruz Biotechnology), anti-IRF9 (Santa Cruz Biotechnology), normal goat IgG (Abcam) and normal rabbit IgG (Cell Signaling Technology) antibodies. ChIP signals were quantified by quantitative PCR analysis with a 7500 real-time PCR system (Applied Biosystems). Values obtained for immunoprecipitated samples (percent (%) input DNA) were normalized to values for respective normal IgG. The specific primer pairs for the Irf5 promoter region and P2rx4 promoter region, respectively, are described below.
Region 1: 5′-ATTTCTCAGGCCCTGTCTAAAGTG-3′ (forward),
5′-GGCACAGAGAGAGTTAGAGGAAGA-3′ (reverse)
Region 2: 5′-TATGGAGTCTTTCTGCACCCTGT-3′ (forward),
5′-TTCCAAGAACGAAGAGTCCCCTA-3′ (reverse)
ISRE-1: 5′-GCTGGCTCGTTTCAAGAATATT-3′ (forward),
5′-CGTACCCTGTAGCCGTCTATT-3′ (reverse)
ISRE-2: 5′-TCTACAGCCTGAAAGTCTATCATTG-3′ (forward),
5′-AAGGAATCTGAGAGGTACACACTG-3′ (reverse)
ISRE-3: 5′-GATAGGGAGAGGCTCGTTCA-3′ (forward),
5′-TAAAAGCTCGGGACCTGGAA-3′ (reverse)
ISRE-4: 5′-TACTGACCTGCCTCTTTTAAGGACA-3′ (forward),
5′-CGGAAAGAACTTTGAACCTTGAG-3′ (reverse)
Region 1: 5′-ATTTCTCAGGCCCTGTCTAAAGTG-3′ (forward),
5′-GGCACAGAGAGAGTTAGAGGAAGA-3′ (reverse)
Region 2: 5′-TATGGAGTCTTTCTGCACCCTGT-3′ (forward),
5′-TTCCAAGAACGAAGAGTCCCCTA-3′ (reverse)
ISRE-1: 5′-GCTGGCTCGTTTCAAGAATATT-3′ (forward),
5′-CGTACCCTGTAGCCGTCTATT-3′ (reverse)
ISRE-2: 5′-TCTACAGCCTGAAAGTCTATCATTG-3′ (forward),
5′-AAGGAATCTGAGAGGTACACACTG-3′ (reverse)
ISRE-3: 5′-GATAGGGAGAGGCTCGTTCA-3′ (forward),
5′-TAAAAGCTCGGGACCTGGAA-3′ (reverse)
ISRE-4: 5′-TACTGACCTGCCTCTTTTAAGGACA-3′ (forward),
5′-CGGAAAGAACTTTGAACCTTGAG-3′ (reverse)
3
Cell Culture and Antibody Validation
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HeLa S3 and HEK293 cells were grown in Dulbecco's modified essential medium supplemented with 10% fetal calf serum. Antibodies used in this study are as follows: Anti-STAT1α/β (sc-346; Santa Cruz Biotechnology), anti-STAT2 (sc-476; Santa Cruz Biotechnology), anti-IRF9 (sc-10793; Santa Cruz Biotechnology), anti-phospho-(Tyr701) STAT1 (#9171; Cell Signaling Technology), anti-phospho-(Tyr689) STAT2 (#07-224; Upstate Biotechnology), anti-phospho-(Ser727) STAT1 (#06-802; Upstate Biotechnology), anti-Pol II (sc-899; Santa Cruz Biotechnology), anti-β-Actin (A5441; SIGMA), anti-Histone H3 (ab1791; Abcam), anti-acetyl-Histone H3 (06-599; Millipore), anti-Histone H1.2 (ab4086; Abcam), anti-Flag (F3165; SIGMA) and anti-TAF-Iα/β (monoclonal antibody KM1725) (34 (link)) antibodies.
4
Antibody Acquisition for Protein Analysis
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Anti-PKR, anti-eiF2, anti-IRF3, anti-IRF9, anti-NF-kB and anti-OAS1 antibodies were obtained from Santa Cruz; the anti-phospho-PKR antibody was obtained from Abcam; anti-phospho-eiF2, anti-STAT1, anti-phospho-STAT1 antibody were obtained from Cell Signaling Technologies; the anti-NS5A antibody (9E10) was generously obtained by Dr. C. Rice; and anti-calnexin antibody was obtained from Sigma.
5
Protein Expression Analysis by Western Blot
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Protein samples were denatured, separated by SDS/PAGE and transferred onto PVDF membrane (Biorad). Membranes were blocked followed by incubation with primary antibodies anti-ZNF677 (1:100, Abcam), anti-IRF9 (1:50, Santa Cruz Biotechnology), anti-ISG15 (1:1000, Cell Signaling), anti-CSH1 (1:375, Thermo Scientific), anti-ACTB (1:200, Abcam) and anti-GAPDH (0.05mg/ml, Sigma Aldrich). Appropriate secondary HRP antibodies were used and membranes were visualized using ECL Western blotting substrate (Thermo Scientific).
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