Figure 1.

Cyclin D3-dependent kinase activities are inversely correlated with the level of p27^kip1 in a cell cycle-dependent manner. To examine whether cyclin D3 activity was regulated during growth transitions, we utilized asynchronous cultures of BALB/c 3T3 cells in growth medium (DMEM) supplemented with 10% CS. Cultures were harvested at 24-h intervals during the growth cycle until density arrest was attained (lanes 1–4). After growth arrest, quiescent cells were mitogenically stimulated with growth medium supplemented with 10% CS + 10 ng/ml PDGF and harvested 18 h later (lane 5). (A) Portions of each cell extract, containing 40 μg of protein, were subjected to immunoprecipitation with cyclin D3 antibody-linked beads. Immunoprecipitates were used to measure in vitro kinase activity using GST-Rb as a substrate. Phosphorylated GST-Rb was separated on 11% SDS-PAGE and visualized by autoradiography. Parallel samples of each cell extract, containing 40 μg of protein, were subjected to Western blot analysis and probed with cyclin D3 antibodies (panel B) and p27^kip1 antibodies (panel C). Proteins were detected using the ECL system as described in MATERIALS AND METHODS.