Micro-quantification of cell-cycle regulating factors by Enzyme-Linked Immuno-Cycling Assay

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Neural cells must develop to mature after free from cell-cycle and never divide again. Despite these facts, cell-cycle regulating factors, i.e.cyclins and cyclin-dependent kinases (cdk) are shown to be exist in post-mitotic neurons. To clarify the physiological roles of those molecules in neuron, we first intended to identify the molecular species of cyclin and cdk in embryonic chick retina. The immunohistochemical studies using cultured retinal neurons revealed that antibodies raised against cyclin D1 or cdk5 were immunoreactive to all the kind of retinal neurons including immature photoreceptor cells. And then next we examined developmental change of cyclin D1 and cdk5 expression in rat cerebellum by immunoblotting. It was found that cyclin D1 was consistently expressed after birth to adulthood, however expression of cdk5 was transient around 8-days-old where the layr formation of cerebellum is extremely remarkable. These results suggest that cdk5 may be involved in the maturation of granule cells in cerebellum. We also found that nitric oxide (NO) is necessary for the maturation and migration of granule cells during the layr formation of the cerebellum. When nitric oxide synthase (NOS) was irreversibly inhibited, migration of granule cells was drastically decreased, and consequently perturbation of the layr formation occurred. To identify the subclass of NOS responsible for this phenomenon, about 1 to 3 mug in dry weight of tissue from external granular layrs, molecular layrs and internal granular layrs was electrophoresed and analyzed by micro-immunoblotting. Micro-analysis revealed that neuronal-type NOS and also endothelial-type NOS were expressed in each layrs during the layr formation.