Cyclin D1 interacts with FOXO1 in vitro and in vivo. (a) Interaction of endogenous cyclin D1 and FOXO1 proteins in LNCaP cells as shown by co-immunoprecipitation using an antibody against cyclin D1. (b) LNCaP cells were transfected with plasmids as indicated. At 48 h after transfection, cells were subjected to immunoprecipitation and western blot analysis. (c) Binding of 35S-labeled cyclin D1 proteins, produced by in vitro transcription and translation, to GST–FOXO1 recombinant proteins. Top, autography of in vitro-produced cyclin D1 proteins bound by GST–FOXO1 recombinant proteins. Middle, input of 35S-labeled cyclin D1 proteins. Bottom, GST and GST–FOXO1 recombinant proteins used for in vitro protein-binding assays indicated by Coomassie blue staining