Ifit1

Manufactured by Cell Signaling Technology

Sourced in United States

IFIT1 is a protein that plays a role in the cellular response to viral infection. It is involved in the interferon signaling pathway and can inhibit viral replication. IFIT1 functions by binding to and inhibiting the translation of viral RNA.

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Rabbit anti-IFIT1 (2 citations)

25 protocols using ifit1

Immunoblotting Analysis of Cell Signaling

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Samples were lysed in RIPA lysis buffer system (Santa Cruz) according to manufacturer’s instructions. 50 µg of protein in cell lysates were separated in 4–20% SDS polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were incubated with the following primary antibodies against IFIT1 (Cell Signaling), IFIT2 (Abcam), IFIT3 (Abcam), cleaved PARP (Cell signaling), BCL2 (Cell signaling) and β-actin (Cell Signaling). Anti-rabbit and anti-mouse IgG antibodies tagged with horseradish peroxidase (Cell signaling) were used. Uncropped scans of the blots are available in Supplementary Figs. 7 and 8.

Mbofung R.M., McKenzie J.A., Malu S., Zhang M., Peng W., Liu C., Kuiatse I., Tieu T., Williams L., Devi S., Ashkin E., Xu C., Huang L., Zhang M., Talukder A.H., Tripathi S.C., Khong H., Satani N., Muller F.L., Roszik J., Heffernan T., Allison J.P., Lizee G., Hanash S.M., Proia D., Amaria R., Davis R.E, & Hwu P. (2017). HSP90 inhibition enhances cancer immunotherapy by upregulating interferon response genes. Nature Communications, 8, 451.

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Immunoblot Analysis of ISG15 Signaling

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Cells were lysed using RIPA buffer supplemented with freshly added protease and phosphatase inhibitors. Protein concentrations were measured by bicinchoninic acid (BCA) assay, equal amounts of protein were separated by SDS‐PAGE, transferred to a nitrocellulose membrane using semi‐dry blotting method, incubated with the respective antibodies, and bands visualized with Amersham enhanced chemiluminescence western blot detection reagent (GE Healthcare Science, Pittsburgh, USA). iNTAS imaging device (iNTAS Science Imaging, Göttingen, Germany) was used for imaging and measurement of relative band intensities. The following antibodies were used to detect the respective proteins: Human ISG15 (sc‐166755, 1:500); pSTAT1 (58D6, 1:1000); IFIT1 (14769S, 1:1000); MX1 (37849S, 1:1000); pAKT (4060S, 1:2000) (Cell Signaling Technology, MA, USA); β‐Actin (ab49900, 1:20 000) (Abcam, Cambridge, UK).

Waqas S.F., Sohail A., Nguyen A.H., Usman A., Ludwig T., Wegner A., Malik M.N., Schuchardt S., Geffers R., Winterhoff M., Merkert S., Martin U., Olmer R., Lachmann N, & Pessler F. (2022). ISG15 deficiency features a complex cellular phenotype that responds to treatment with itaconate and derivatives. Clinical and Translational Medicine, 12(7), e931.

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Interferon Signaling Pathway Protein Detection

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The antibodies used were IRF-1 (8478S, Cell Signaling), IFIT1 (14769S, Cell Signaling), IFIT3 (sc-393512, Santa Cruz Biotechnology), Mx1 (37849S, Cell Signaling), ISG15 (2758T, Cell Signaling), IFI44L (HK7931, Hushi Pharmaceutical Technology), Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb (9733, Cell Signaling), Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb (9751, Cell Signaling), anti-rabbit IgG, HRP-linked antibody (7074P2, Cell Signaling), anti-mouse IgG, HRP-linked antibody (7076P2, Cell Signaling). Anti-A/California/7/2009-like HA serum (sheep 606 and 610) (14/310) was purchased from National Institute for Biological Standards and Control (NIBSC). Anti-sheep IgG, HRP-linked antibody (F030231) was purchased from Beijing BioRab Technology. Mouse anti-hnRNP U monoclonal antibody (ab10297) was purchased from Abcam.
Polyinosinic-polycytidylic acid sodium salt (P9582) was purchased from Sigma-Aldrich. Human IFN-β (10704-H02H) was purchased from Sino Biological. MRT67307 HCl (T5162) and Pyrrolidinedithiocarbamate ammonium (T3147) were purchased from TargetMol.

Zhao L., Xia M., Wang K., Lai C., Fan H., Gu H., Yang P, & Wang X. (2020). A Long Non-coding RNA IVRPIE Promotes Host Antiviral Immune Responses Through Regulating Interferon β1 and ISG Expression. Frontiers in Microbiology, 11, 260.

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Immunoblot Analysis of Osimertinib Response

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For immunoblot analysis, cells were plated at 250,000 cells per 10-cm dish. After 24 h, cells were treated with 300 nM osimertinib or DMSO control for 3 days. Cells were collected in phosphate-buffered saline, centrifuged (3 min at 3000 rpm), and suspended in MAP Kinase Lysis Buffer. Aliquots of the cell lysates containing 50 µg of protein were submitted to SDS-PAGE on 8% polyacrylamide gels, transferred to nitrocellulose, and immunoblotted. All primary antibodies were used at a dilution of 1:1000 in 3% BSA in 1× TBST. The following antibodies were used: PARP (Cell Signaling Technology 9542S), IFIT1(Cell Signaling Technology 14796S), MX2 (Abcam 22479), STAT1(Cell Signaling Technology 9172S), and ß-actin (Cell Signaling Technology 4967S). The immunoblots were derived from the same experiment processed in parallel.

Gurule N.J., McCoach C.E., Hinz T.K., Merrick D.T., Van Bokhoven A., Kim J., Patil T., Calhoun J., Nemenoff R.A., Tan A.C., Doebele R.C, & Heasley L.E. (2021). A tyrosine kinase inhibitor-induced interferon response positively associates with clinical response in EGFR-mutant lung cancer. NPJ Precision Oncology, 5, 41.

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Immunoblotting of cell signaling proteins

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Proteins were resolved on a 10% SDS–PAGE, transferred to polyvinylidene difluoride filters (Immobilon-P; Millipore), and probed with primary antibodies, including Phospho-Tyr701-STAT1 (# 7649), Phospho-Ser536-p65 (# 3031), IRF1 (# 8478), IFIT1 (# 12082), P-ERK1/2 (# 9101), ERK1/2 (# 9102) all from Cell Signalling Technology (Beverly, MA, USA); STAT1 (# sc-346), STAT2 (# sc-476), IRF9 (# sc-10793), from Santa Cruz Biotechnology (Inc., CA, USA); IFN-κ (# H00056832-M01, clone 1B7, Abnova GmbH, Heidelberg, Germany). Anti-actin and anti-histone 4 (H4) antibodies (both from Santa Cruz) were used for loading control of total cell and nuclear cell lysates, respectively.

Lulli D., Carbone M.L, & Pastore S. (2016). Epidermal growth factor receptor inhibitors trigger a type I interferon response in human skin. Oncotarget, 7(30), 47777-47793.

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Protein Immunoblotting Analysis Protocol

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Proteins were analyzed according to our previously described protocol [15 (link)]. The primary antibodies were the following: IRF1 (#8478), phospho-Tyr701-STAT1 (#7649), phospho-ERK1/2 (#9101), ERK1/2 (#9102), IFIT1 (#12082), all from Cell Signalling Technology (Beverly, MA, USA); STAT1 (#sc-346) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); IFN-κ (#H00056832-M01, clone 1B7) from Abnova GmbH (Heidelberg, Germany). Anti-actin antibody (#sc-1615, Santa Cruz Biotech.) was used for loading control of total cell lysates.

Lulli D., Carbone M.L, & Pastore S. (2017). The MEK Inhibitors Trametinib and Cobimetinib Induce a Type I Interferon Response in Human Keratinocytes. International Journal of Molecular Sciences, 18(10), 2227.

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Inflammatory Signaling Pathway Analysis

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Menthol and bacterial lipopolysaccharide (LPS) (Escherichia coli, 0111: B4) were purchased from Sigma (USA). Mass spectrometry grade reagents (column, buffer et al.) were all purchased from Sigma (USA). The Mouse Proteome Profiler Array was purchased from R&D Systems (USA). The antibodies used were CCL3 (ab25128, Abcam, USA), IL-6 (12912; Cell Signaling Technology, USA), TNF-α (11948S; Cell Signaling Technology, USA), p-NF-κB (3033; Cell Signaling Technology, USA), NF-κB (8242; Cell Signaling Technology, USA), p-Akt (4051; Cell Signaling Technology, USA), Akt (9272; Cell Signaling Technology, USA), TLR1(2209; Cell Signaling Technology, USA), TNFAIP3 (5630; Cell Signaling Technology, USA), IFIT1 (14769; Cell Signaling Technology, USA), viperin (13996; Cell Signaling Technology, USA), IL-1β (AF-401-NA, R&D Systems, USA), and β-actin (A1978; Sigma-Aldrich, USA). HRP-conjugated secondary antibodies were obtained from Cell Signaling Technologies (7074, 7076; USA).

Shahid M., Lee M.Y., Yeon A., Cho E., Sairam V., Valdiviez L., You S, & Kim J. (2018). Menthol, a unique urinary volatile compound, is associated with chronic inflammation in interstitial cystitis. Scientific Reports, 8, 10859.

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STAT1/2 Activation and Antiviral Effectors in ZIKV-Infected Cells

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STAT1 and STAT2 signaling was studied in A549 cells as previously described [61 (link)]. Briefly, A549 cells were infected with the indicated ZIKV strain at an MOI of 0.1 and 1 (based on Vero cell titration). At 48hpi, cells were pulse treated with 1000 IU/mL of recombinant human IFNβ (PBL Assay Science) for 30 minutes and whole-cell lysates were collected in modified radioimmunoprecipitation assay buffer supplemented with Halt Protease Inhibitor Cocktail (ThermoFisher) and phosphatase inhibitor cocktail II (Calbiochem). Western blot analysis was performed to detect STAT1 phosphotyrosine residue 701 (Cell Signaling), total STAT1 (Cell Signaling), STAT2 phosphotyrosine residue 689 (Upstate, EMD Milipore), total STAT2 (Cell Signaling), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cell Signaling). Protein expression levels were quantified using Image Lab software. For analysis of antiviral effector proteins within human moDCs, 4e5 cells were used per condition and protein lysates were collected as described for A549 cells. The following antibodies were obtained from Cell Signaling: RIG-I, MDA5, LGP2, STAT1, STAT2, IFIT1, viperin, and GAPDH. The IFIT3 antibody was kindly provided by Dr. G. Sen.

Bowen J.R., Quicke K.M., Maddur M.S., O’Neal J.T., McDonald C.E., Fedorova N.B., Puri V., Shabman R.S., Pulendran B, & Suthar M.S. (2017). Zika Virus Antagonizes Type I Interferon Responses during Infection of Human Dendritic Cells. PLoS Pathogens, 13(2), e1006164.

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SARS-CoV-2 Nucleocapsid Protein Detection

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Human cell lines were grown to 70% confluence and lysed in 1X RIPA buffer (50mM Tris-HCl pH 7.4, 150mM NaCl, 1mM EDTA, 0.1% SDS, 1% NP-40, 0.25% sodium deoxycholate) supplemented with EDTA-free protease inhibitor cocktail (Roche). Proteins were separated by SDS-PAGE on 4–12% Bis-Tris gels, transferred to a nitrocellulose membranes, blocked in Intercept (PBS) blocking buffer (LI-COR) or 5% milk, and incubated with primary antibodies overnight at 4°C. Washed membranes were incubated for 45 min to 1 hour at room temperature in secondary antibody solution (LI-COR IRDye 680, 800; 1:10,000 in 5% milk or Intercept (PBS) blocking buffer), imaged on an Odyssey^® CLx scanner, and analyzed using the Image Studio Software. Antibodies were used at the following dilutions: ACE2 (R&D Systems #AF933, 1:200), β-actin (Sigma #A5316, 1:5000 or Santa Cruz Biotech #sc8432, 1:500), Vinculin (Santa Cruz #sc-73614, 1:2000), p STAT1-Y701 (Cell Signaling #9167, 1:1000), p STAT1-S727 (Cell Signaling #8826, 1:1000), STAT1 (Cell Signaling #14994, 1:1000), MX1 (Cell Signaling #37849, 1:1000), IFIT1 (Cell Signaling #14769, 1:1000), STING (Cell Signaling #13647S, 1:1000), cGAS (Cell Signaling #15102S, 1:1000), TBK1 (Cell Signaling #38066S, 1:1000), IRF3 (Cell Signaling #4302S, 1:1000), SARS-CoV-2 Nucleocapsid (Sino Biological # 40588-T62, 1;1000 or # 40143-MM05, 1:1000).

Puray-Chavez M., LaPak K.M., Jasuja R., Pan J., Xu J., Eschbach J.E., Mohammed S., Lawson D.Q., Wang Q., Brody S.L., Major M.B., Goldfarb D, & Kutluay S.B. (2024). A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models. bioRxiv.

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Immunoblotting of Interferon-Stimulated Proteins

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Immunoblotting/Western blotting was performed as previously described (13 , 14 (link)). Membranes were probed with primary antibodies recognizing total PR (Santa Cruz Biotechnology, sc-7208 or ThermoScientific, MS-298-P), IRF9 (Cell Signaling, 76684), IFIT1 (Cell Signaling, 14769), IFIT3 (Santa Cruz Biotechnology, sc-393512), OAS1 (Cell Signaling, 14498), and ERK (Cell Signaling, 9102). All Western blotting experiments were performed in triplicate, and representative experiments are shown.

Walter K.R., Goodman M.L., Singhal H., Hall J.A., Li T., Holloran S.M., Trinca G.M., Gibson K.A., Jin V.X., Greene G.L, & Hagan C.R. (2017). Interferon-Stimulated Genes are Transcriptionally Repressed by PR in Breast Cancer. Molecular cancer research : MCR, 15(10), 1331-1340.

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