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. 2016 Aug 22;291(43):22607–22617. doi: 10.1074/jbc.M116.727081

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© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 4. — HCV NS5A forms a ternary complex with Abi1 and Sos1. A, HEK293T cells were cotransfected with FLAG-tagged Abi1, Myc-tagged NS5A, and HA-tagged Sos1. At 48 h after transfection, cell lysates were immunoprecipitated (IP) with an anti-HA antibody and bound proteins were immunoblotted with an anti-Myc antibody or an anti-FLAG antibody. Input corresponds to 10% of total proteins as indicated. B, HEK293T cells were transfected with either FLAG-tagged Abi1 or HA-tagged Sos1, or cotransfected with both constructs as indicated. Total cell lysates harvested at 24 h after transfection were incubated with either purified GST- or GST-NS5A-conjugated glutathione beads. Bound proteins were detected by immunoblot analysis with either anti-HA or anti-FLAG antibodies. Protein expression levels of GST and GST-NS5A were verified by using an anti-GST antibody. The arrow indicates GST-NS5A fusion protein. Experiments were performed in duplicate.