Isoform-specific cyclin E regulation by Fbw7. (A) Lysates from two independent clones of each Hct116 genotype were analyzed for cyclin E abundance and kinase activity (Grb2, loading control). (B) Pulse-chase analysis of cyclin E in Hct116 knockout lines. Cells were labeled 1 h after release from aphidicolin arrest (see text). Quantitation of the phosphorimage is depicted in the graph, and the primary data for each isoform-specific cell line is shown. (C) HeLa cells and 293A cells were transfected with the indicated Fbw7 siRNAs (α, β, γ, and CR) or control siRNAs (GFP and C). Cyclin E abundance, kinase activity, and T62 phosphorylation were measured 72 h after transfection. (D) 293A cells and HeLa cells were cotransfected with a FLAG-Fbw7γ expression vector in combination with the indicated siRNAs (γ-tubulin, loading control). (E) 293A cells were transfected with vectors expressing myc-tagged wild-type or P382I cyclin E. Lysates were immunoprecipitated with anti-Myc tag antibody (9E10), and cyclin E abundance, kinase activity, and T62 phosphorylation are shown.