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. 2023 Jan 20;299(3):102930. doi: 10.1016/j.jbc.2023.102930

Figure 1.

Figure 1

Expression of SARS-CoV-2 ORF8 and interaction with complement C3 and its metabolites by Coimmunoprecipitation.A, multimeric forms of ORF8-FLAG–tagged protein, expressed in transfected HepG2 cells and immunoblotted using anti-FLAG antibody in lysates prepared under reduced (R) versus nonreduced (NR) condition. Monomers (17 kDa) in standard Laemmli buffer, oligomerizes in NR condition. Co-IP from FLAG-ORF8–transfected HepG2 cells as indicated in panels. B, IP with mouse-anti-FLAG antibody and detection on blot by rabbit anti-C3 antibody against C3dg/TED domain encompassing fragment (amino acid 1000–1326 of human C3); C, IP with rabbit anti-C3 antibody and detection on blot with anti-FLAG. D, reprobing of the stripped-blot in C panel with rabbit anti-C3. Anti-C3 recognizes epitopes in C3, C3b, iC3b, and C3c fragments, as indicated. Antibody light chain (LC) and heavy chain (HC) are also shown. For control lanes (anti-rabbit IgG antibody), IP lanes (target antibodies), and input lanes (lysate), 15% equivalent and 5% of total input samples were loaded, respectively. Data shown is representative image of three independent experiments. The colorimetric images for prestained markers (Blueye prestained protein marker; GeneDireX) in panels and the Chemiluminescence images from the corresponding immunoblots were captured and merged using Image Lab software (Bio-Rad), associated with ChemiDoc XRS+ imaging system (Bio-Rad) to generate the representative images.