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ISG15 is a protein involved in the post-translational modification of other proteins. It functions as a ubiquitin-like modifier, which can be conjugated to target proteins, altering their function, interactions, or subcellular localization.

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Lab products found in correlation

Cxcl10, by Thermo Fisher Scientific (2 mentions) Iscript cdna synthesis kit, by Bio-Rad (2 mentions) Il 10, by Thermo Fisher Scientific (2 mentions) Nbp1 89300, by Novus Biologicals (1 mentions) Vinculin, by Cell Signaling Technology (1 mentions) Rig 1, by Cell Signaling Technology (1 mentions) Sting, by Cell Signaling Technology (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) β actin, by Merck Group (1 mentions) A1978, by Merck Group (1 mentions) Ifna4, by Thermo Fisher Scientific (1 mentions) Taqman gene expression master mix, by Thermo Fisher Scientific (1 mentions) Ddx58, by Thermo Fisher Scientific (1 mentions) Ifna1, by Thermo Fisher Scientific (1 mentions) Cxcl9, by Thermo Fisher Scientific (1 mentions) Ifit1, by Thermo Fisher Scientific (1 mentions) Gapdh, by Thermo Fisher Scientific (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Muramyl dipeptide mdp, by InvivoGen (1 mentions) Il 1β, by Thermo Fisher Scientific (1 mentions)

5 protocols using isg15

1

Immunoblotting Protocol for Protein Analysis

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Immunoblotting was performed with antibodies against the following proteins: ISG15 (1:1,000; mouse, 703132, clone 1H9L21; human and rat, PA5-88262; Thermo Fisher Scientific), GAPDH (1:1,000; 2188, Cell Signaling Technology), cGAS (1:1,000; 31659, Cell Signaling Technology), STING (1:1,000; 13647, Cell Signaling Technology), RIG-I (1:1,000; 3743, Cell Signaling Technology), MAVS (1:1,000; 4983, Cell Signaling Technology), β-actin (1:10,000; A1978, MilliporeSigma), filamin-C (1:1,000; NBP1-89300, Novus Biologicals), vinculin (1:1,000; 4650, Cell Signaling Technology), p62 (1:1,000; 109012, Abcam), and LC3 (1:1,000; 12741, Cell Signaling Technology). Immunoblotting for the soluble and insoluble fractions of filamin-C was performed in mouse hearts as previously described (82 (link)). Densitometry was performed using ImageJ version 1.39 (NIH).
Yerra V.G., Batchu S.N., Kaur H., Kabir M.G., Liu Y., Advani S.L., Tran D.T., Sadeghian S., Sedrak P., Billia F., Kuzmanov U., Gramolini A.O., Qasrawi D.O., Petrotchenko E.V., Borchers C.H., Connelly K.A, & Advani A. (2023). Pressure overload induces ISG15 to facilitate adverse ventricular remodeling and promote heart failure. The Journal of Clinical Investigation, 133(9), e161453.
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2

Quantitative PCR analysis of human liver

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Whole liver RNA was isolated using RNeasy mini kit (Qiagen, Hilden, Germany) including DNAse treatment according to manufacturer’s protocol starting with homogenization of liver tissue in RLT buffer. cDNA was generated by using an iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s protocol. Human specific gene expression was measured using Taqman primer/probe quantitative PCR, in TaqMan® Gene Expression Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). Primer/probe combinations were purchased from Thermo Fisher Scientific; CXCL10 (Hs01124251_g1), CXCL9 (Hs00171065_m1), DDX58 (Hs01061436_m1), GAPDH (Hs00266705_g1), IFIT1 (Hs01911452_s1), ISG15 (Hs01921425_s1), IFNA1 (Hs00855471_g1), IFNA4 (Hs01681284_sh), IFNB1 (Hs01077958_s1), MX1 (Hs00895608_m1), OAS1 (Hs00973637_m1), RSAD2 (Hs00369813_m1), STAT1 (Hs01013996_m1), TLR3 (Hs01551078_m1). Expression of target genes was normalized to the expression of GAPDH using the formula 2−ΔCt, ΔCt = Cttarget−CtGADPH. cDNA from non-chimeric mouse livers was used as control to test cross-reactivity of housekeeping and target genes. Due to the difference in hepatocyte donor baseline expression levels of examined genes (Suppl. Fig. 1), fold changes of transcripts were calculated to those of non-infected humanized livers from mice transplanted with the identical hepatocyte donor.
van de Garde M.D., Pas S.D., van Oord G.W., Gama L., Choi Y., de Man R.A., Boonstra A, & Vanwolleghem T. (2017). Interferon-alpha treatment rapidly clears Hepatitis E virus infection in humanized mice. Scientific Reports, 7, 8267.
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3

Optimized Immune Modulation Assays

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The PRR-ligands used were: the lipopeptide Pam3CysSK4 [P3C] [TLR2/1] [300 ng/mL] [#L2000, EMC microcollections], Lipomannan [LM] [TLR2/6] [30 ng/mL] [#tlrl-hkmt-1], polyinosinic:polycytidylic acid (poly[I:C) [TLR3] [5–70 µg/mL] [#tlrl-pic], lipopolysaccharide [LPS] [TLR4] [100 ng/mL] [#tlrl-peklps], Flagellin [TLR5] [100 ng/mL] [#tlrl-stfla], the antiviral compound R848 [TLR7/8] [100 ng/mL] [#tlrl-r848], the peptidoglycan component muramyl dipeptide [MDP] [NOD2] [1 µg/mL] [#tlrl-mdp], all from InvivoGen, and unmethylated CpG dinucleotides [TLR9] [10 μM] [# 1712649, TibMolBiol].
Recombinant cytokines used were: IL-10 [100 ng/mL] [#200–10], IL-1β [100 ng/mL] [#200-01B], TNF [100–200 ng/mL] [#300-01A], and IFNβ [0.1–10 ng/ml] [#300-02BC] [all from PeproTech], IL12 [20 ng/mL] [#219-IL, R&D], and ISG15 [500 ng/ml] [#12729HNAE, Thermo Fisher Scientific]. JAK-inhibitors used were: filgotinib [10 µM] [GLPG0634] [#S7605, Selleckchem.com] and ruxolitinib [10 µM] [#tlrl-rux, InvivoGen]. Neutralising antibodies used were: rabbit anti-human IFN-β [2.5 µg/ml] [#500-P32B, PeproTech] and isotype control rabbit IgG [2.5 µg/ml] [#X0903, Dako Cytomation]. For details, see Supplementary Table 1, available as Supplementary data at ECCO-JCC online.
Østvik A.E., Svendsen T.D., Granlund A.V., Doseth B., Skovdahl H.K., Bakke I., Thorsvik S., Afroz W., Walaas G.A., Mollnes T.E., Gustafsson B.I., Sandvik A.K, & Bruland T. (2020). Intestinal Epithelial Cells Express Immunomodulatory ISG15 During Active Ulcerative Colitis and Crohn’s Disease. Journal of Crohn's & Colitis, 14(7), 920-934.
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4

RT-qPCR Analysis of Antiviral Genes

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RNA was extracted using TRIzol Reagent (Invitrogen) and cDNA was generated with the iScript cDNA synthesis kit (Bio-Rad). TaqMan primer/probe sets for murine Setdb2, Ifna2, Ifnb1, Irf3, Irf7, Ccl2, Cxcl1, Cxcl2, Il10, Tnf, Il10, Tnf, Pkr, Rnasel, Isg15, Mx1, and human SETDB2 were purchased from Applied Biosystems. NS1 and M1 were detected using custom primers [5 (link)]. Gene expression was assessed using an ABI Prism 7500 instrument (Applied Biosystems) and normalized to Gapdh or ACTB.
Kroetz D.N., Allen R.M., Schaller M.A., Cavallaro C., Ito T, & Kunkel S.L. (2015). Type I Interferon Induced Epigenetic Regulation of Macrophages Suppresses Innate and Adaptive Immunity in Acute Respiratory Viral Infection. PLoS Pathogens, 11(12), e1005338.
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5

RNA Purification and qPCR for Viral Genes

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The RNA purification and qPCR was preformed as described11 (link), with the following primers from Applied Biosystems: TLR3 (Mm01207404_m1), cGAS (Mm01147497_m1), STING (Mm01158117_m1), MX1 (Mm00487796_m1), ISG15 (Mm01705338_s1), Viperin (Mm00491265_m1), IL-6 (Mm00446190_m1), IFN-β (Mm00439552_s1), CXCL10 (Mm00445235_m1), HSV-1 gB (the forward primer (5′-CGC ATC AAG ACC ACC TCC TC-3′), the reverse primer (5′-AGC TTG CGG GCC TCG TT-3′) and probe: 5′-CGG CCC AAC ATA TCG TTG ACA TGG C-3′), Iba-1 (Mm00479862_g1) and HSV-1 LAT (The forward primer (5′-ACCCACGTACTCCAAGAAGGC-3′), the reverse primer (5′-TAAGACCCAAGCATAGAGAGCCA-3′ and probe (5′-TCCCACCCCGCCTGTGTTTTTGT-3′)). Levels of mRNAs of interest were normalized to β-actin using the formula 2Ct(βactin)−Ct(mRNA X). The resulting normalized ratio were either presented directly in the figures or further normalized to the normalized ratio of untreated WT cells as specified in the figure legends.
Reinert L.S., Lopušná K., Winther H., Sun C., Thomsen M.K., Nandakumar R., Mogensen T.H., Meyer M., Vægter C., Nyengaard J.R., Fitzgerald K.A, & Paludan S.R. (2016). Sensing of HSV-1 by the cGAS–STING pathway in microglia orchestrates antiviral defence in the CNS. Nature Communications, 7, 13348.
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