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USP18 is a laboratory reagent produced by Cell Signaling Technology. It is an enzyme that plays a role in the regulation of cellular signaling pathways.

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Lab products found in correlation

Stat2, by Merck Group (6 mentions) Stat1, by Santa Cruz Biotechnology (6 mentions) Ripa buffer, by Thermo Fisher Scientific (6 mentions) Phospho tyr 701 stat1, by Cell Signaling Technology (5 mentions) β actin, by Cell Signaling Technology (5 mentions) Isg15, by Santa Cruz Biotechnology (5 mentions) Ifit1, by Cell Signaling Technology (5 mentions) Phospho tyr 689 stat2, by Merck Group (4 mentions) Protease phosphatase inhibitor cocktail, by Cell Signaling Technology (4 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions) Pierce ecl western blotting substrate, by Thermo Fisher Scientific (3 mentions) Gapdh, by Merck Group (2 mentions) β actin, by ABclonal (2 mentions) Ra1 lysis buffer, by Macherey-Nagel (2 mentions) Horseradish peroxidase, by Abcam (1 mentions) Stat2, by Santa Cruz Biotechnology (1 mentions) Mabf227, by Abcam (1 mentions) Py stat1, by BD (1 mentions) Hrp conjugated rabbit igg, by Abcam (1 mentions) Hrp conjugated mouse igg, by Abcam (1 mentions)

10 protocols using usp18

1

Immunoblotting Analysis of IFNλ4 Signaling

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The cells were lysed with RIPA buffer (Thermo Fisher Scientific) to prepare total cell lysates. Ten micrograms of each cell lysate were loaded on SDS-PAGE gels prior to immunoblotting. The antibodies used for immunoblotting were: IFNλ4 (1:200, mouse, Millipore MABF227), IFNλ4 (1:200, rabbit, Abcam ab196984), STAT1 (1:1000, rabbit, BD Biosciences 610120), PY-STAT1 (1:1000, mouse, BD Biosciences 612233), STAT2 (1:1000, rabbit, Santa Cruz Biotechnology sc-476), IRF9 (1:1000, rabbit, Santa Cruz sc-496), SOCS1 (Abcam #62584), USP18 (Cell Signaling Technology #4813), horseradish peroxidase (HRP)-conjugated rabbit IgG (1:5000, Abcam ab97051), and HRP-conjugated mouse IgG (1:5000, Abcam ab97023).
Chung J.H., Hong S.H., Seo N., Kim T.S., An H.J., Lee P., Shin E.C, & Kim H.M. (2019). Structure-based glycoengineering of interferon lambda 4 enhances its productivity and anti-viral potency. Cytokine, 125, 154833.
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2

Immunoblotting and Immunoprecipitation Workflow

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Whole-cell extracts for immunoblotting were prepared by incubating cells for 10 min in RIPA lysis buffer (Thermo Fisher Scientific) with 50 mM dithiothreitol and Protease/Phophatase inhibitor cocktail (Cell Signaling Technology). For coimmunoprecipitation assay, cells were lysed in 50 mM Tris, pH 6.8, 0.5% NP-40, 200 mM NaCl, 10% glycerol, 1 mM EDTA, and 1× Protease/Phophatase inhibitor cocktail (Cell Signaling Technology). Cell lysates were incubated with V5 conjugated with protein G dynabeads for 2 h at room temperature. Immunoprecipitates were subjected to Western blotting. Immunoblotting was performed using the Bio-Rad Western blot workflow. Membranes were blocked in 5% BSA for primary antibodies or 5% nonfat dry milk for secondary antibodies. Antibodies used were STAT1 (Santa Cruz Biotechnology), STAT2 (Millipore), phospho-Tyr 701 STAT1 (Cell Signaling Technology), phospho-Tyr 689 STAT2 (Millipore), USP18 (Cell Signaling Technology), β-actin (ABclonal), and GAPDH (Millipore). Signal was detected with enhanced chemiluminescence detection reagent (SuperSignal West pico; Thermo Fisher Scientific) by film development.
Gruber C., Martin-Fernandez M., Ailal F., Qiu X., Taft J., Altman J., Rosain J., Buta S., Bousfiha A., Casanova J.L., Bustamante J, & Bogunovic D. (2020). Homozygous STAT2 gain-of-function mutation by loss of USP18 activity in a patient with type I interferonopathy. The Journal of Experimental Medicine, 217(5), e20192319.
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3

Immunoblotting Protocol for Protein Analysis

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Cells were lysed, and immunoblotting was performed as previously described (Bianco and Mohr, 2017 (link)). The antibodies used in this study were as follows: TIF-IA (Bethyl Laboratories; A303-065A) UBF (Santa Cruz; SC-13125), Akt (Cell Signaling; 9272), Fibrillarin (Santa Cruz; sc-166001), UL44 (Virusys; CA006), pp28 (Virusys; CA004-100), IE1/IE2 (Millipore; MAB810), HMGB2 (Cell Signaling Technology; 14163), USP18 (Cell Signaling Technology; 4813), IFIT2 (Proteintech; 12604–1-AP), TFIIIA (Bethyl; A303-621A), ISG15 (Proteintech; 15981–1-AP), RIG-I (Proteintech; 20566), MDA5 (Proteintech; 21775–1-AP), UBA7 (Cell Signaling Technology; 69023), actin (Cell Signaling Technology; 3700), BrdU (Sigma; B2531), p53 (Cell Signaling Technology; 9282).
Bianco C, & Mohr I. (2019). Ribosome biogenesis restricts innate immune responses to virus infection and DNA. eLife, 8, e49551.
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4

Western Blot Analysis of Cellular Proteins

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Cell lysates were placed in RIPA buffer with the Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Lysates were size-fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Membranes were independently probed with indicated antibodies and visualized using Clarity Western ECL 10 Substrate (Bio-Rad). Primary antibodies were: ATGL (#2138; Cell Signaling Technology), USP18 (#4813; Cell Signaling Technology), β-actin (#3700; Cell Signaling Technology), HA tag (#3724S, Cell Signaling Technology), UBE1L (#61266, Cell Signaling Technology), Tubulin, UCP1 (#PA1–24894, ThermoFisher Scientific), Myc-tag (#2276S, Cell Signaling Technology), and ISG15 (#2743, Cell Signaling Technology). Secondary antibodies were Goat anti-rabbit IgG (#170–6515; Bio-Rad) or Goat anti-mouse IgG (#170–6516; Bio-Rad).
Liu X., Lu Y., Chen Z., Liu X., Hu W., Zheng L., Chen Y., Kurie J.M., Shi M., Mustachio L.M., Adresson T., Fox S., Roszik J., Kawakami M., Freemantle S, & Dmitrovsky E. (2020). The Ubiquitin Specific Protease USP18 Promotes Lipolysis, Fatty Acid Oxidation and Lung Cancer Growth. Molecular cancer research : MCR, 19(4), 667-677.
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5

Western Blot Analysis of Cellular Proteins

Cited 1 time
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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 described55 (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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6

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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7

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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8

Immunoblotting of JAK-STAT Signaling Proteins

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Whole-cell extracts for immunoblotting were prepared by incubating cells for 20 min in RIPA lysis buffer (Thermo Fisher Scientific) with 50 mM dithiothreitol and Protease/Phosphatase inhibitor cocktail (Cell Signaling Technology). Protein was quantified with Micro BCA Protein Assay (Thermo Fisher Scientific). Immunoblotting was performed using the Bio-Rad Western blot workflow. Membranes were blocked in 5% BSA for primary antibodies or 5% nonfat dry milk for secondary antibodies. Antibodies used were STAT1 (Santa Cruz Biotechnology), STAT2 (Millipore), phospho-Tyr 701 STAT1 (Cell Signaling Technology), phospho-Tyr 689 STAT2 (Cell Signaling Technology), USP18 (Cell Signaling Technology), IFIT1 (Cell Signaling Technology), MX1 (Abcam), SOCS1 (Thermo Fisher Scientific), β-actin (ABclonal), and GAPDH (Millipore). Signal was detected with enhanced chemiluminescence detection reagent (ECL or SuperSignal West pico, Thermo Fisher Scientific) by film development or capturing on ImageQuant 800 Fluor imaging system (Cytiva). For all representative immunoblots shown, n=3 separate experiments were carried out.
Malle L., Martin-Fernandez M., Buta S., Richardson A., Bush D, & Bogunovic D. (2022). Excessive Negative Regulation of Type I Interferon Disrupts Viral Control in Individuals with Down Syndrome. Immunity, 55(11), 2074-2084.e5.
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9

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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10

Immunoblotting and Coimmunoprecipitation Assays

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For immunoblotting, cells were lysed in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific), Dithiothreitol, and a protease/phosphatase inhibitor cocktail (Cell Signaling Technology). For coimmunoprecipitation assay, cells were lysed in 50 mM Tris, pH 6.8, 0.5% NP-40, 200 mM NaCl, 10% glycerol, 1 mM EDTA, and 1× protease/phosphatase inhibitor cocktail (Cell Signaling Technology). Cell lysates were incubated with Flag-M2 beads for 2 h at room temperature or with V5 antibodies for 2 h at 4°C, then incubated with Protein A Sepharose 4 Fast Flow and Protein G Sepharose 4 Fast Flow beads (GE Healthcare) for 1 h at 4°C. Immunoprecipitates were subjected to Western blotting. The antibodies used were directed against STAT1 (Santa Cruz Biotechnology), STAT2 (Millipore), phospho-Tyr 689 STAT2 (Millipore), ISG15 (Santa Cruz Biotechnology), mAb cl.2.1, a gift of E.J. Borden, Cleveland Clinic, Cleveland, OH), V5 (Invitrogen), and Flag (Sigma-Aldrich). Antibodies to phospho-Tyr 701 STAT1, USP18, β-Actin, IFIT1, and AKT were all from Cell Signaling Technology. The chemiluminescence detection reagent was Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) or Western Lightning Plus-ECL (PerkinElmer).
Martin-Fernandez M., Buta S., Le Voyer T., Li Z., Dynesen L.T., Vuillier F., Franklin L., Ailal F., Muglia Amancio A., Malle L., Gruber C., Benhsaien I., Altman J., Taft J., Deswarte C., Roynard M., Nieto-Patlan A., Moriya K., Rosain J., Boddaert N., Bousfiha A., Crow Y.J., Jankovic D., Sher A., Casanova J.L., Pellegrini S., Bustamante J, & Bogunovic D. (2022). A partial form of inherited human USP18 deficiency underlies infection and inflammation. The Journal of Experimental Medicine, 219(4), e20211273.
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