FIG. 3.

Aging liver increases activities of cdk4 and cdk6 by elevation of cyclin D3. A and B. Protein levels of cyclin D3 are increased in old livers. Nuclear extracts from five livers of young and old animals were examined by Western blotting with antibodies to cyclin D3. The membrane was reprobed with Abs to β-actin. Coomassie stain shows staining of a parallel gel loaded with the same proteins. Bar graphs (B) represent a summary of multiple experiments. Levels of cyclin D3 were calculated as a ratio to β-actin. C. C/EBPα is observed in complexes with cyclin D3 in the liver. Cyclin D3 was immunoprecipitated from young and old livers, and cdk4, cdk6, and C/EBPα were examined by Western blotting in cyclin D3 IPs. IgG lanes, heavy chains of immunoglobulin G were detected by Coomassie staining of the membrane. An aliquot from each reaction mixture was incubated with GST-C/EBPα substrate in the presence of [γ-³²P]ATP (kinase assay, bottom part). D. cdk4-cyclin D3 are associated with the C/EBPα-Brm complex in old livers. Nuclear extracts from young and old livers were fractionated by size exclusion chromatography on an SEC-400 column as described in the legend to Fig. 1. Positions of molecular mass markers are shown on the top. Western lanes: examination of cdk4, cdk6, cyclin D3, and C/EBPα in the gel filtration fractions by Western blotting. C/EBPα-IP lanes, C/EBPα was immunoprecipitated from fractions and C/EBPα IPs were probed with antibodies to cyclin D3. Kinase assay lanes: cdk4 and C/EBPα were immunoprecipitated from each fraction and examined in a kinase assay with GST-Rb and with GST-C/EBPα substrates. Positions of cyclin D3-cdk4/cdk6 complexes are shown on the bottom.