In vitro reconstitution of Cyclin E ubiquitylation by a RhoBTB3^CUL3/RBX1 ubiquitin ligase complex. (A) RhoBTB3 depletion in HEK293T cells assayed by immunoblot; Golgin 97 was used as a loading control. (B) Characterization of anti-FLAG immunopurified complexes containing Myc-RhoBTB3, FLAG-CUL3, and HA-RBX1 proteins; asterisks indicate a nonspecific band. (C) Coomassie blue gel and immunoblot analysis of purified FLAG–Cyclin E1 (lane 1) and FLAG–CyclinE1–CDK2-HA complex (lane 2) isolated by immunoprecipitation from HEK293T cells. (D) In vitro–reconstituted ubiquitylation of Cyclin E using complexes shown in B. Left columns, anti–Cyclin E immunoblots; right columns, anti-ubiquitin immunoblots. Top gel, 6% to show larger species; bottom gel, 7.5% to include Cyclin E. Quantitation of lanes 1–7 of the bottom gels gave relative values of: 0.05, 0.06, 0.05, 1, 0.16, 0.09, and 0.06 (left); 0, 0, 0, 1, 0.1, 0.05, and 0 (right). (E) In vitro ubiquitylation of Cyclin E or Cyclin E–CDK2 complex as in D; top panel represents a long exposure of the same gel shown below (blotted using anti–Cyclin E). At the bottom is shown a blot for CDK2-HA in the same samples. Quantitation of lanes 1–7 gave relative values of: 0.3, 0.3, 0.3, 1, 0.4, 0.1, and 0 (long exposure). Molecular mass marker mobility is shown at the left (A–C) or right (D and E) in kilodaltons.