RhoBTB3-mediated regulation of Cyclin E ubiquitylation and stability in vivo. (A) Immunoblot detection of Cyclin E1 ubiquitylation in HeLa cells treated with control or RhoBTB3-siRNA #1. After 48 h, cells were transfected with FLAG–CyclinE1 and HA-ubiquitin; after 72 h total, extracts were precipitated with anti-FLAG antibody, eluted with 0.2 mg/ml FLAG peptide, and blotted using anti-HA (left panel) or anti–Cyclin E (right panels). The far right panel is a longer exposure (Long exp.) of the middle panel (Short exp.). Bottom, immunoprecipitated FLAG–Cyclin E1 and input (5%). (B) Exogenous RhoBTB3 expression increases Cyclin E1 ubiquitylation. Analysis was as in A; RhoBTB3 was transfected in HeLa cells 12 h before other components, and samples were collected 24 h later. Asterisks indicate IgG heavy chain. (C) Determination of Cyclin E stability. U2OS cells transfected for 72 h with either control or RhoBTB3 siRNA were treated with 50 µg/ml cycloheximide as indicated and Cyclin E levels determined by immunoblotting. Graph shows mean Cyclin E level for each time point (circles) from at least two independent experiments; error bars represent SEM. (D) Synchronized HeLa cells (Hengst et al., 1994) were analyzed for their content of the proteins indicated at right, monitored by immunoblot at the indicated times after release of thymidine-nocodazole block. A representative example from one of two independent experiments is shown. Graph represents quantitation of the blots below. Molecular mass marker mobility is shown at the right (A and B) or left (D) in kilodaltons.