Fig. 4.
Effect of PACAP on mitosis and cyclin E/CDK2 kinase activity and protein levels in cortical precursor culture. Top, PACAP inhibited DNA synthesis by 35% in 8 hr cortical cultures. [3H]thymidine was added at 5 hr, and incorporation was assayed at 8 hr. Data are expressed as mean cpm ± SEM. *p < 0.05. Middle, Using parallel cultures, extracts (30 μg per sample) of control and PACAP (10−8m)-treated cells were analyzed for CDK2 kinase activity, using methods as in Figure 2. PACAP decreased CDK2 activity by ∼75%. Hela cells were used as positive control [(+)CON]. Extracts were also subjected to Western blot analysis to determine whether PACAP regulated CDK2 protein levels. No change was observed in total CDK2 protein levels after PACAP treatment. Immunoblots were reprobed with actin antibody to verify equal protein loading. Experiments were performed three times yielding similar results. Bottom, Parallel extracts were analyzed for cyclin E protein levels by immunoblotting, indicating no change, a result obtained in four separate experiments.