Unphosphorylated Cyclin E is a substrate of recombinant RhoBTB3^CUL3/RBX1 in vitro. (A) CUL3–RBX1 binary complexes (lane 1 in Coomassie blue gel and immunoblot) were purified from E. coli coexpressing both GST-CUL3 and HA-RBX1. Assembly of RhoBTB3^CUL3/RBX1 complex was performed by mixing CUL3–RBX1 with purified FLAG-RhoBTB3 bound to anti-FLAG antibody–conjugated agarose. Coomassie blue staining and immunoblot analysis of ternary complexes eluted with 0.2 mg/ml FLAG peptide are shown in lane 2 of gel and blot. Asterisks indicate IgG heavy and light chains. (B) Coomassie blue staining and immunoblot analysis of GST–Cyclin E1 purified with glutathione-Sepharose from E. coli (lane 1 in gel and blot). (C) Time course of RhoBTB3^CUL3/RBX1 -dependent ubiquitylation of GST–Cyclin E1 using recombinant complexes shown in A; mRhoBTB3–CUL3–RBX1 or bRhoBTB3–CUL3–RBX1 complexes were purified from mammalian cells or bacteria, respectively; bCUL3–RBX1 binary complex was purified from bacteria. The presence of RhoBTB3 in each reaction (middle blot) was detected using anti-RhoBTB3 antibody. As a control, bottom blot shows similar levels of RBX1 present in the 40-min reactions. Molecular mass marker mobility is shown at left (A and B) or right (C) in kilodaltons.