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. 2014 Nov 19;290(1):296–309. doi: 10.1074/jbc.M114.560169

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FIGURE 2. — The UBA domain of SCCRO binds to polyubiquitinated CRL substrates. A, GST-SCCRO pulldown products from HeLa cell lysate pretreated with MG132 were released by thrombin cleavage. SCCRO and SCCRO-interacting proteins were further immunoprecipitated with antibody against polyubiquitinated proteins and analyzed by Western blotting with antibodies against Aurora B and RHOA (CUL3 substrates) and cyclin E and p27 (CUL1 substrates), which showed that SCCRO selectively bound to polyubiquitinated Aurora B and RHOA. B, experiments were performed as described for A except that HeLa cells were transfected with Aurora B or RHOA before MG132 treatment. Note that GST-SCCRO, but not GST or GST-SCCRO^L30A, bound to polyubiquitinated Aurora B and RHOA. IP, immunoprecipitation; IB, immunoblotting; PD, pulldown.