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3 protocols using anti ifitm1

1

Immunological Antibody Characterization Protocol

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The table below describes the commercial antibodies used, their catalog numbers, and their company.
Western blot and immunofluorescence antibodies.
Anti-Alix (sc-49267)(Santa Cruz Biotechnology)
Anti-β-actin (MA1-91399)(Thermo Fisher Scientific)
Anti-β-actin (sc-47778)(Santa Cruz Biotechnology)
Anti-CD4 (2009-09)(Novocastra)
Anti-CD63 (sc-15363)(Santa Cruz Biotechnology)
Anti-EEA1 (610456)(BD Biosciences)
Anti-Flotillin1 (ab41927)(Abcan)
Anti-GAPDH (G9545)(Sigma-Aldrich)
Anti-GFPGift from R. Hedge (MRC)
Anti-ΗΑ (H3663)(Sigma-Aldrich)
Anti-HLA-A (15240-1-AP)(Proteintech)
Anti-IFITM1 (60074-1-Ig)(Proteintech)
Anti-IFITM2 (66137-1-Ig)(Proteintech)
Anti-IFITM3/2 (11714-1-AP)(Proteintech)
Anti-Lat1 (sc-54229)(Santa Cruz Biotechnology)
Anti-TfR (136800)(Thermo Fisher Scientific)
Anti-Tsg101 (976126)(BD Bioscencies)
Anti-Nef (2949)(NIH AIDS Reagent Program)
Anti-Neurexin1 (ab77596)(BD Bioscencies)
Anti-Syntenin1 (ab133267)(Abcan)
Anti-IgG Mouse-HRP(GE)
Anti-IgG Rabbit-HRP(GE)
Anti-IgG Goat-HRP(GE)
Anti-IgG Mouse-Alexa 594(Thermo Fisher Scientific)
Anti-IgG Rabbit-Alexa 647(Thermo Fisher Scientific)
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2

Interferon Signaling Pathway Analysis

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Human IFN-β recombinant protein (rhIFN-β) and human IFN-λ1 recombinant protein (rhIFN-λ1) were purchased from Abbkine (Wuhan, China) and MedChemExpress (Shanghai, China). The Cell Counting Kit (CCK-8) was obtained from Biosharp (Anhui, China). Pyridone 6 (JAK inhibitor I) (A13457) was purchased from Adooq Biosciences (Irvine, CA, USA). Antibodies used for Western blotting, anti-phospho-STAT1 (S727), anti-STAT1, and anti-IFI35 antibodies (all rabbit polyclonal antibodies), were obtained from Abmart (Shanghai, China). Anti-IFIT2, anti-TLR3, anti-IRF7, anti-IRF3, anti-USP18, and anti-ISG15 antibodies (all rabbit) were purchased from ABclonal (Wuhan, Hubei, China). Anti-JAK1, anti-JAK2, anti-phospho-JAK1, and anti-phospho-JAK2 antibodies (all rabbit) were purchased from Abcam (Cambridge, MA, USA). Anti-MDA5 (IFIH1) and anti phospho-IRF3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-RIG-I (DDX58), anti-IFIT1, anti-IFIT3, anti-IFIT5, anti-IRF1, anti-IFITM1, and anti-β-actin antibodies were purchased from Proteintech (Chicago, IL, USA), and the antibodies used are listed in Additional file 7: Table S7. The monoclonal antibody against the JEV envelope (E) was kindly provided by Shengbo Cao (Huazhong Agricultural University, Wuhan, China). For immunofluorescence, rabbit anti-FLAG antibody was purchased from Proteintech.
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3

Western Blot Analysis of IFITM Proteins

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Whole-cell lysates were prepared with Laemmeli buffer and resolved by SDS-PAGE. The samples were then transferred to Immobilon-P membrane (Millipore) and probed with the indicated antibodies. Anti-IFITM3 anti-sera (Abgent, AP1153a) was used at 1:250. Anti-IFITM3 from Abcam (Anti-Fragilis (ab15592)) was used at 1:500. Anti-IFITM1 (Proteintech, catalog no. 60074-1-Ig) was used at 1:1,000. Actin (Sigma, catalog no. A2228-200UL) was used at 1:5,000. Secondary anti-Rabbit HRP conjugated antibody (Jackson Immuno Research, 211-062-171) and anti-mouse HRP conjugated antibody (Jackson Immuno Research, 115-035-003) were used at 1:10,000.
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