Fig. 4

Cyclin D3 interacted with actin in vivo and in vitro. a MCF-7 or MDA-MB231 cells were transfected with HA-actin. 48 h later, cells were lysed and immunoprecipitated with HA antibody or Cyclin D3 antibody, then subjected to SDS-PAGE and detected with Cyclin D3 antibody or HA antibody. b GST-actin (70KD) was in vitro translated, [35S]methionine labeled, preimmobilized onto glutathione-Sepharose 4B beads, and incubated with lyses of MCF-7 cells transfected with Cyclin D3 (30KD) or Cyclin D1 (30KD). Binding proteins were subjected to SDS-PAGE and visualized by phosphorimaging. c MCF-7 cells were subjected to immunoflurorescent staining assay. Cells were fixed and reacted with a mouse monoclonal anti-Cyclin D3 antibody and a rabbit polyclonal anti-actin antibody. The secondary antibodies were anti-rabbit IgG-conjugated to fluorescein isothiocyanate and anti-mouse IgG-conjugated to rhodamine red. The images were captured with a Leica confocal microscope and software provided by Leica