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. 2011 Aug 2;286(38):33613–33621. doi: 10.1074/jbc.M111.245126

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© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 2. — Cul3-KLHL7 is an E3 ligase. A, FLAG-KLHL7 or mock vectors were co-expressed with HA-Ub in HCT116 cells and treated with 20 μm MG132 (+) or DMSO (−) prior to harvesting. Cell lysates were immunoprecipitated (IP) with anti-FLAG antibody and blotted with anti-FLAG and anti-HA antibodies. B, FLAG-KLHL7 was co-expressed with HA-Ub in HCT116 cells. After 20 μm MG132 treatment, the cell lysates were immunoprecipitated with anti-HA antibody and subjected to immunoblotting (IB) with anti-HA and anti-FLAG antibodies. C, in vitro ubiquitination assay using Cul3-KLHL7 complex immunopurified from HCT116 cells. Cul3-KLHL7 was incubated with recombinant E1 (UBE1), E2 (UBC4), and Ub, with or without ATP at 30 °C. The reaction was stopped at the indicated times and then immunoblotted with anti-FK2 Ub antibody.