RhoBTB3 interacts with free Cyclin E. (A) HEK293T cells were transfected with the indicated constructs and immunoprecipitated with llama anti-GFP antibody. Samples were analyzed by immunoblot with mouse anti-Myc antibody (left portion) or rabbit anti-GFP (right portion). A Ponceau S–stained gel is shown at the bottom left. (B) Quantitation of RhoBTB3 in anti-Cyclin immunoprecipitates, normalized to inputs. *, P < 0.05; error bars represent SEM, n ≥ 3. (C) Anti-FLAG immunoprecipitation of endogenous Cyclin E in cells transfected with FLAG-RhoBTB3. The input image was from a different exposure but confirms equal loading of the left and right lanes. (D) Schematic representation of RhoBTB3 and derived constructs. Bottom: cells were cotransfected with the indicated constructs and either Cyclin A2–GFP or Cyclin E1–GFP; immunoprecipitation was performed as in A. (E) Cells cotransfected with FLAG-RhoBTB3, Myc–Cyclin E1, or CDK2-HA were analyzed by immunoprecipitation and blotting as in C. Molecular mass marker mobility is shown at the left in kilodaltons.