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.
Anti irf1
Manufactured by Santa Cruz Biotechnology
Sourced in Japan, United Kingdom
Anti-IRF1 is a laboratory reagent that specifically binds to and detects the Interferon Regulatory Factor 1 (IRF1) protein. IRF1 is a transcription factor that plays a key role in the regulation of genes involved in immune and inflammatory responses. The Anti-IRF1 product can be used in various research applications, such as Western blotting, immunoprecipitation, and immunohistochemistry, to investigate the expression and localization of IRF1 in different biological systems.
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Lab products found in correlation
Anti β actin, by Merck Group (3 mentions)
Anti stat1, by Cell Signaling Technology (2 mentions)
Anti irf8, by Santa Cruz Biotechnology (2 mentions)
Anti cdk4, by Santa Cruz Biotechnology (2 mentions)
Anti stat1, by Santa Cruz Biotechnology (2 mentions)
Anti ido1, by Abcam (1 mentions)
Cd4 microbeads, by Miltenyi Biotec (1 mentions)
Anti β actin, by Abcam (1 mentions)
Normal goat igg, by Abcam (1 mentions)
Normal rabbit igg, by Cell Signaling Technology (1 mentions)
Anti irf9, by Santa Cruz Biotechnology (1 mentions)
Anti irf7, by Santa Cruz Biotechnology (1 mentions)
Anti irf5, by Abcam (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Anti irf3, by Santa Cruz Biotechnology (1 mentions)
Simplechip enzymatic chromatin ip kit, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Anti rnap 2, by Santa Cruz Biotechnology (1 mentions)
Chemidoc analyzer, by Bio-Rad (1 mentions)
Anti phospho tyr701 stat 1, by Cell Signaling Technology (1 mentions)
10 protocols using anti irf1
1
Isolation and Culture of Hepatic and Immune Cell Populations
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
Western Blot Analysis of Cellular Proteins
4
IFNγ-Induced Protein Analysis in BMDMs
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BMDMs (106 cells/well) were stimulated with IFNγ (100 U/ml) for the times indicated, lysed and used for Western blot analysis as described previously (30 (link)) with the following adaptations: cells were lysed in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% IGEPAL CA-630 (v/v), 10% glycerol (v/v), 0.1 mM EDTA, 2 mM DTT, 0.2 mM Na-vanadate, 25 mM Na-fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 0.1 μg pepstatin and 1 mM PMSF. The following antibodies were used: anti-IRF1 (Santa Cruz, SC-640), anti-phospho-Tyr701 STAT1, and anti-STAT1 (Cell Signaling Technology, 9167 and 9172), anti-pan-ERK (BD Transduction Laboratories, 610123; p42 is shown in our experiments). Peroxidase-conjugated secondary antibodies (mouse and rabbit) were from Cell Signaling Technology (7076 and 7074). Blots were scanned with a Chemidoc analyzer (Bio-Rad).
5
Visualization of PD-L1 Promoter Regulation
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The assay for PD-L1 promoter visualization was performed as previously described38 (link). In brief, 20 sgRNAs (sgARRAY) around the PD-L1 promoter were cloned into a PUC19 backbone via Golden Gate reaction. The sgARRAY was verified via EcoRI digest. pTETON-dCas9-24*GCN4, pTETon-scFv-GCN4-sfGFP and sgARRAY were cotransfected into 143B cells for 6 h, and 0.5 μg ml–1 doxycycline was added into the medium. Eighteen hours later, cells were treated with 100 U ml–1 IFNγ for another 6 h. Afterward, cells were prepared for immunofluorescence staining. Primary antibodies were anti-KAT8 (Atlas, HPA066324) and anti-IRF1 (Santa Cruz, sc-74530). Secondary antibodies were anti-rabbit Alexa Fluor 594 (Invitrogen, A32754) and anti-mouse Alexa Fluor 405 (Invitrogen, A-31553). SIM analysis was performed using the N-SIM S superresolution microscope (Nikon). The sequences of the sgARRAY are provided in Supplementary Table 8 .
6
Molecular Mechanisms in Cervical Cancer
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Human cervical carcinoma cell lines (CaSki and SiHa) and human embryonic kidney 293T (HEK293T) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained in monolayer cultures according to ATCC recommendations. HUVECs were purchased from Clonetics (Walkersville, MD, USA), and seeded on 0.3% gelatin-coated dishes (Sigma, St. Louis, MO, USA) using the EGM-2 BulletKit medium (Clonetics). Wortmannin and LY294002, which are PI3K inhibitors, were obtained from Sigma (St. Louis, MO, USA). The following antibodies were used in this study: anti-HPV16 E6 (ab226447), anti-IgG (Abcam, Cambridge, UK), anti-IRF-1, anti-Flag, anti-cyclin D1, anti-CDK4, anti-NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Bax, anti-Bcl-xL, anti-Bcl-2, anti-p53, anti-VEGFR-1, anti-VEGFR-2, anti-phospho-VEGFR-2(Tyr-1175), anti-HIF-1α, anti-PI3K, anti-phospho-PI3K, anti-Akt, anti-phospho-Akt(Ser473), anti-PDK-1, anti-phospho-PDK-1(Ser241), anti-mTOR, anti-phospho-mTOR(Ser2448), anti-4E-BP1, anti-phospho-4E-BP1(Thr70) (Cell Signalling, Beverly, MA, USA), anti-p21, anti-VEGF (Ab-1; Oncogene, Cambridge, MA, USA), and anti-β-actin (Sigma, St. Louis, MO, USA).
7
Western Blotting of Tumor Proteins
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Proteins from tumour cell lysates were separated by SDS–polyacrylamide gel electrophoresis, blotted on nitrocellulose membranes and probed with the following primary antibodies: anti-STAT1 (Santa Cruz, clone M-22, 1:1000) and anti-pSTAT1 (Cell Signaling, clone 58D6, 1:1,000), anti-IRF1 (Santa Cruz, clone H-205, 1:500) and anti-GAPDH (Cell Signaling, 14C10, 1:5,000). HC10 (1:1,000) was used for detection of β2m-free HLA heavy chains58 (link)59 (link). After washing, membranes were incubated with the appropriate secondary antibodies linked to horseradish peroxidase. Antibody binding was visualized with the enhanced chemiluminescence (ECL) system. Full scans of western blots are depicted in Supplementary Figs 7–9 .
8
Confocal Microscopy of moDC Responses
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For confocal laser scanning microscopy, 105 moDC were incubated at 37°C for 2 h on 6 channel μ-Slide (Ibidi) and subsequently stimulated for 4 h with 107 M. stadtmanae cells. After fixation in 3% paraformaldehyde, primary antibodies were added at 1:100 in PBS with 3% BSA and 0.1% saponin and incubated overnight at 4°C. Cells were incubated with anti-NF-κB p65 (1:100, F-6), anti-IRF1 (1:100, B-1), or anti-IRF5 (1:100, H-56, all from Santa Cruz Biotechnology) overnight at 4°C, followed by staining with an Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L) secondary antibody (1:300, Invitrogen). The nuclei were counterstained using Hoechst 33342 dye (1:3,000, Life Technologies). For detection of ASC specks, BLaER1 cells were stimulated for 4 and 18 h, and staining was performed as previously described. ASC (1:100, B-3, Santa Cruz Biotechnology) was used as the primary antibody, and Alexa Fluor 546-conjugated goat anti-mouse IgG (H + L) was used as the secondary antibody (1:300, Invitrogen). Images were captured using the TCS SP5 confocal microscope and LAS AF software (both from Leica).
9
Enterocyte Isolation and Molecular Analysis
10
ChIP-seq Analysis of Th17 Cell Transcription Factors
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