Figure 4.

STAT-5B mediates PDGF-BB-induced cyclin D1 expression, CDK4 activity, and Rb protein phosphorylation in VSMCs. A: The conditions were the same as in Figure 3 except that cells were transduced with either Ad-GFP (control) or Ad-dnSTAT-5B with a MOI of 80 and quiesced. Cells were then treated with and without PDGF-BB (20 ng/ml) for 16 hours and analyzed for cyclin D1 expression, CDK4 activity, Rb protein phosphorylation, and CDK4 levels as described above in Figure 3. The bar graph at the bottom represents the quantitative analysis of three independent experiments. B: Nuclear extracts were prepared from quiescent control and various times of PDGF-BB (20 ng/ml)-treated VSMCs, and an equal amount of protein from each condition was analyzed for DNA binding activity using a ³²P-labeled putative STAT-binding sequence from rat cyclin D1 promoter as a probe. To detect the presence of STAT-5B in the protein-DNA complexes, anti-STAT-5B antibodies (2 μg) were added to the reaction mix and incubated for an additional 3 hours on ice. C: ChIP was performed with control and various time periods of PDGF-BB-treated VSMCs using anti-STAT-5B antibodies, and the resulting DNA fragments were subjected to PCR amplification using primers spanning −1013 to −411 region of rat cyclin D1 promoter. *P < 0.01 versus control; **P < 0.01 versus PDGF-BB treatment alone.