Isg15

Manufactured by Santa Cruz Biotechnology

Sourced in Germany

ISG15 is a small protein that is induced in response to viral infection or type I interferon stimulation. It functions as an ubiquitin-like modifier and is involved in the regulation of immune and inflammatory responses.

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11 protocols using isg15

Protein Analysis by Immunoblotting

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Cell pellets were lysed and sonicated in EBC lysis buffer (50 mM Tris-Cl, pH7.4, 120mM NaCl, 0.5% NP-40, 1mM EDTA) containing HALT Protease and Phosphatase Inhibitor cocktail (Thermo Scientific) and 1mM phenylmethylsulfonyl fluoride (PMSF). Thirty μg of protein were separated on SDS-polyacrylamide gels. Proteins were transferred to PVDF (Millipore) and probed with antibodies. ARF (mouse), Actin, p53 (human), Gamma-tubulin, H-Ras, ISG15 (human), and STAT1 were all purchased from Santa Cruz Biotechnologies. p53 (mouse), phospho-STAT1^Tyr701, phospho-STAT1^Ser727, phospho-STAT3^Tyr705, and STAT3 were purchased from Cell Signaling. ARF (human) and GAPDH were purchased from Bethyl Laboratories, and MDM2 was from Millipore. The mouse ISG15 antibody was a gift from Dr. Deborah Lenschow. Secondary horseradish peroxidase conjugated antibodies (Jackson Immunoresearch) were used and ECL plus was used to visualize the bands (GE Healthcare).

Forys J.T., Kuzmicki C.E., Saporita A.J., Winkeler C.L., Maggi LB J.r, & Weber J.D. (2014). ARF and p53 coordinate tumor suppression of an oncogenic IFN-β-STAT1-ISG15 signaling axis. Cell reports, 7(2), 514-526.

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Comprehensive Immunoblot Profiling

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IRGM (Abcam # 69494; 1:500), NOD1 (CST# 3545S; 1:1000), NOD2 (Proteintech # 20980‐1‐AP; 1:1000), RIPK2 (CST#4142S; 1:1000), Anti‐beta Actin (Abcam # ab6276; 1:5000), GFP (Abcam # ab290; 1:5000), Flag M2 (Sigma # F3165; 1:1000), p62 (BD Biosciences # BD‐610832; 1:1000), ATG5 (CST # 2630S; 1:1000), c‐Myc (Santa Cruz # sc‐40; 1:1000), HA‐Tag (CST # 3724S; 1:1000), RICK (Santa Cruz # sc‐22,763; 1:1000), Anti‐LC‐3B antibody (Sigma # L7543; 1:1000), Anti‐RICK A‐10 (Santa Cruz # sc‐166,765; 1:1000), IL‐1β (CST#12242), Anti‐pro Caspase1 + p10 + p12 Antibody (Abcam # ab179515; 1:1000), ISG15 (Santa Cruz# sc‐166755; 1:500), Phospho‐p38 MAPK(Thr180/Tyr182; 1:1000; CST#9211; 1:1000), Phospho‐NF‐κB p65 (ser536) (93H1) (CST# 3033; 1:1000). HRP conjugate secondary antibodies were purchased from Novus (1:2000) or Promega (1:5000).

Mehto S., Jena K.K., Yadav R., Priyadarsini S., Samal P., Krishna S., Dhar K., Jain A., Chauhan N.R., Murmu K.C., Bal R., Sahu R., Jaiswal P., Sahoo B.S., Patnaik S., Kufer T.A., Rusten T.E., Chauhan S., Prasad P, & Chauhan S. (2022). Selective autophagy of RIPosomes maintains innate immune homeostasis during bacterial infection. The EMBO Journal, 41(23), e111289.

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Western Blot Analysis of Cellular Proteins

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Cells were lysed in a modified lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 50 mM NaF, 10 mM β-glycerophosphate, 10 nM calyculin A, 1 mM Na3VO4, and protease inhibitors), followed by SDS-PAGE and western blotting analysis as previously described^{55 (link)}. The primary antibodies against STAG2 (Santa Cruz, SC-81852), IRF9 (Cell Signaling, #76684), IRF7 (Cell Signaling, #4920), USP18 (Cell Signaling, #4813), ISG15 (Santa Cruz, SC-166755), IRF1 (Cell Signaling, #8478), IRF3 (Cell Signaling, #11904), GAPDH (Cell Signaling, #2118), GAPDH (Cell Signaling, #51332), STAG1 (Novus Biologicals, NB100-298), PD-L1 (R&D Systems, AF156), CTCF (Cell Signaling, #2899), and Flag M2 (Sigma, F3165) were used. All primary antibodies were used at a 1:1,000 dilution, with the exception of GAPDH (1:3000) and PD-L1 (1:200).

Chu Z., Gu L., Hu Y., Zhang X., Li M., Chen J., Teng D., Huang M., Shen C.H., Cai L., Yoshida T., Qi Y., Niu Z., Feng A., Geng S., Frederick D.T., Specht E., Piris A., Sullivan R.J., Flaherty K.T., Boland G.M., Georgopoulos K., Liu D., Shi Y, & Zheng B. (2022). STAG2 regulates interferon signaling in melanoma via enhancer loop reprogramming. Nature Communications, 13, 1859.

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Immunoblotting for JAK-STAT Signaling

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Cells were lysed in radioimmunoprecipitation-assay buffer, and protein extract was used for Western blotting. The antibodies that were used were directed against STAT1 (Santa Cruz Biotechnology), STAT2 (Millipore), phospho-Tyr701 STAT1 (Cell Signaling Technology), phosphor-Tyr689 STAT2 (Millipore), USP18 (Cell Signaling Technology), ISG15 (Santa Cruz Biotechnology), β-actin (Cell Signaling Technology), and IFIT1 (Cell Signaling Technology).

Alsohime F., Martin–Fernandez M., Temsah M., Alabdulhafid M., Le Voyer T., Alghamdi M., Qiu X., Alotaibi N., Alkahtani A., Buta S., Jouanguy E., Al–Eyadhy A., Gruber C., Hasan G.M., Bashiri F.A., Halwani R., Hassan H.H., Al–Muhsen S., Alkhamis N., Alsum Z., Casanova J., Bustamante J., Bogunovic D, & Alangari A.A. (2020). JAK Inhibitor Therapy in a Child with Inherited USP18 Deficiency. The New England journal of medicine, 382(3), 256-265.

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Antibodies for Western Blot Analysis

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ADRP/Perilipin 2 (#15294-1-AP), DGAT1 rabbit polyclonal antibodies (#11561-1-AP) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mouse monoclonal antibodies (#60004-1-Ig; ProteinTech Group, Chicago, IL), Calnexin rabbit polyclonal antibodies (#ADI-SPA-865; Enzo Life Sciences, Lörrach, Germany), HBsAg (20-HR20) rabbit polyclonal antibodies (#20-HR20; Fitzgerald Industries, Acton, MA), PLIN3 guinea pig polyclonal antibodies (#GP30s; Progen, Heidelberg, Germany), ISG15 (#sc-166755; Santa Cruz Biotechnology, Heidelberg, Germany), and Oas1a mouse monoclonal antibodies (#sc-365072; Santa Cruz Biotechnology) were used according to the manufacturer’s protocols. Anti-LHBs mouse monoclonal antibodies (Virology, Giessen, Germany) were described previously.⁵² (link)

Churin Y., Irungbam K., Imiela C.S., Schwarz D., Mollenkopf H.J., Drebber U., Odenthal M., Pak O., Huber M., Glebe D., Roderfeld M, & Roeb E. (2021). Lipid Storage and Interferon Response Determine the Phenotype of Ground Glass Hepatocytes in Mice and Humans. Cellular and Molecular Gastroenterology and Hepatology, 12(2), 383-394.

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Western Blot and qPCR Analysis of Cellular Signaling

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For the experiment shown in Figure 2, cells were lysed in RIPA buffer (Thermo Fisher Scientific), DTT and a protease/phosphatase inhibitor cocktail (Cell Signaling Technology) and subjected to western blotting. For the experiment shown in Figure 5, cells were harvested in RA1 lysis buffer (Macherey-Nagel, Düren, Germany) containing 1% β-mercaptoethanol. The resulting cell lysates were stored at −20°C until RNA extraction and qPCR analysis. The antibodies used were directed against STAT1 (Santa Cruz Biotechnology), STAT2 (Millipore), phospho-Tyr 701 STAT1 (Cell Signaling Technology), phospho-Tyr 689 STAT2 (Millipore), USP18 (Cell Signaling Technology), ISG15 (Santa Cruz Biotechnology), β-actin (Cell Signaling Technology), and IFIT1 (Cell Signaling Technology). An enhanced chemiluminescence detection reagent was used for detection (Pierce ECL Western Blotting Substrate, Thermo Fisher Scientific).

Martin-Fernandez M., García-Morato M.B., Gruber C., Loza S.M., Malik M.N., Alsohime F., Alakeel A., Valdez R., Buta S., Buda G., Marti M.A., Larralde M., Boisson B., Rodriguez M.F., Qiu X., Chrabieh M., Ayed M.A., Muhsen S.A., Desai J.V., Ferre E.M., Rosenzweig S.D., Amador-Borrero B., Bravo-Gallego L.Y., Olmer R., Merkert S., Bret M., Sood A.K., Al-rabiaah A., Temsah M.H., Halwani R., Hernandez M., Pessler F., Casanova J.L., Bustamante J., Lionakis M.S, & Bogunovic D. (2020). Systemic Type I IFN Inflammation in Human ISG15 Deficiency Leads to Necrotizing Skin Lesions. Cell reports, 31(6), 107633.

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Western Blot Analysis of ISG15 Expression

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Cell lysis and protein extraction was carried out using the RIPA buffer [1 M Tris–HCl pH 8 (PanReac AppliChem, ITW Reagents), 0.5 M EDTA (Thermo Fisher Scientific), Triton™ X-100 (Sigma-Aldrich), 10% sodium deoxycholate (Sigma-Aldrich), 10% SDS (Sigma-Aldrich) and 3 M NaCl (Thermo Fisher Scientific)], supplemented with protease and phosphatase inhibition cocktails (Sigma-Aldrich). 20 µg protein sample were separated by SDS-PAGE, then transferred to 0.2 µm pore-size nitrocellulose membranes (Bio-Rad). Membranes were blocked for 1 h using 5% BSA (PanReac AppliChem, ITW Reagents), in 1X TBS-T (0.1% Tween20, Bio-Rad). Subsequently, the following antibodies were incubated in the same buffer for 16 h at 4 ºC: ISG15 (Santa Cruz Biotechnology, sc-166755), α-tubulin (Sigma-Aldrich, T9026). Membranes were washed with 1X TBS-T, then incubated with Rabbit Anti-Mouse IgG–Peroxidase antibody (Sigma-Aldrich) or Goat Anti-Rabbit IgG H&L peroxidase-conjugated antibody (Abcam, Cambridge, UK). HRP substrate was used for chemiluminescent detection and image acquisition was performed using a Chemidoc Imaging System (Bio-Rad). ISG15 expression was relativised against INT-SFT values.

Mondaza-Hernandez J.L., Moura D.S., Lopez-Alvarez M., Sanchez-Bustos P., Blanco-Alcaina E., Castilla-Ramirez C., Collini P., Merino-Garcia J., Zamora J., Carrillo-Garcia J., Maestro R., Hindi N., Garcia-Foncillas J, & Martin-Broto J. (2022). ISG15 as a prognostic biomarker in solitary fibrous tumour. Cellular and Molecular Life Sciences, 79(8), 434.

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Western Blot for ADAR1, ISG15 and Tubulin

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Medium was aspired from the culture and cells were lysed directly using 2x loading buffer (125mM Tris-Cl, 3% SDS, 10% gylcerol, 0.01% bromphenol blue, 3.3% β-mercaptoethanol). Lysates were boiled for 10 min prior loading on 12% SDS PAGE Tris-Gly gel. Proteins were blotted to PVDF membrane (Bio-Rad) and probed for ADAR1 (Sigma-Aldrich), ISG15 (Santa Cruz) and tubulin (AbCam). Blots were developed using chemiluminiscent detection. All antibodies used for western blot analysis are listed in Supplemental Table S16.

Roučová K., Vopálenský V., Mašek T., Del Llano E., Provazník J., Landry J.J.M., Azevedo N., Ehler E., Beneš V, & Pospíšek M. (2024). Loss of ADAR1 protein induces changes in small RNA landscape in hepatocytes. RNA (New York, N.Y.), 30(9).

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Cell Culture and Antibody Details

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HeLa, HEK293T, and CtBP1^−/− MEFs were maintained in DMEM containing 10% FBS and antibiotic–antimycotic solution. In the case of CtBP1^−/− MEFs, 2 mM L-glutamine was added. All cells were grown in incubators with conditions at 37°C, 5% CO₂, and 95% humidity. The manufacturers and catalog numbers of the antibodies used are as follows: CtBP1 (Santa Cruz, sc-17759), ISG15 (Santa Cruz, sc-166755), Myc (Santa Cruz, sc-40), HA (Cell signaling, #3724), Flag (Sigma, F3165), GFP (Proteintech, 66002), HDAC1 (Santa Cruz, sc-81598), HDAC4 (Proteintech 17449-1-AP), β-actin (Sigma–Aldrich, A1978).

Lim Y.H., Park Y.J., Lee J, & Kim J.H. (2024). Transcriptional corepressor activity of CtBP1 is regulated by ISG15 modification. Animal Cells and Systems, 28(1), 66-74.

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Western Blot and qPCR Analysis of Cellular Signaling

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