FIGURE 7. PKCδ activation by bFGF is required for the activation of AP-1, a direct downstream transcriptional regulatory factor of MAPK signaling.

A, cis-acting control plasmid, pAP-1 Luc, which contains seven tandem repeats of AP-1 consensus sequences was transiently transfected (2 μg per reaction) into human primary adult articular chondrocytes with and without bFGF (100 ng/ml) and in the presence or absence of a general inhibitor of PKC, BIM, or selective inhibitors of PKCδ or PKCα/β. The luciferase activity was calculated by comparing with the luciferase activity of a control plasmid, pAP-1 Luc (assigned as 1). A Renilla vector (100 ng per reaction) was co-transfected as an internal control for normalization. The data represent the means of three independent experiments in duplicate. B, serum-starved cells (>16 h) in monolayer that were stimulated with and without bFGF (100 ng/ml) for 45 min were subjected to nuclear preparation and incubated with biotin-labeled double-stranded AP-1 consensus sequences. Shifted AP-1 protein-DNA complex band was visualized by chemiluminescence imaging. Unlabeled double-stranded AP-1 oligonucleotides were used for competition assays to determine the binding specificity of the assay.