To test this computational prediction, next we performed Kinase assays with Cdk4, Cdk2 and Cdk5 in presence and absence of the R428 inhibitors 8A and 8B. First, we performed Cdk4 kinase assay using different doses of these two compounds, 8A and 8B to see the inhibitory potentials of these compounds on Cdk4 activity. We found that these compounds, 8A and 8B significantly blocked kinase activity of Cdk4 at the concentrations that provided neuroprotective effects i.e 5 mM and 1 mM respectively. Moreover, kinase assays with other Cdks such as Cdk2 and Cdk5 indicated that these inhibitors specifically inhibited Cdk4 and did not block the kinase activity of Cdk2 and Cdk5. We have also checked the endogenous Cdk4 kinase activity after NGF deprivation in presence and absence of the inhibitors 8A and 8B. We immunoprecipitated Cdk4 from total cell lysates of treated and untreated differentiated PC12 cells, then the immunoprecipitated protein was subjected to kinase assay. Results showed about two-fold increase in Cdk4 kinase activity following NGF deprivation in neuronal PC12 cells and that activity was almost completely blocked in presence of the inhibitors. Taken together, our results suggest that these inhibitors are specific to Cdk4 and they are capable of blocking the NGF AMN107 deprivationinduced increase of Cdk4 activity. Rb proteins directly bind with E2F proteins and actively repress expression of E2F responsive genes in live neurons. Upon phosphorylation by Cdk4 it translocates out from nucleus to cytosol in response to certain apoptotic stimuli. Phosphorylation of Rb proteins results in dissociation of E2F-Rb repressor complex on E2F responsive pro-apoptotic genes thereby induces expression of those genes. In response to NGF deprivation, Rb proteins are phosphorylated due to activation of Cdk4. We determined the phosphorylation levels of Rb protein in neuronal PC12 cells after NGF deprivation in presence or absence of Cdk4 inhibitors. Immunocytochemical staining followed by fluorescence imaging studies reveal that intensity of the phospho- Rb staining which is mostly present in cytosol is greatly increased after NGF deprivation and that level is significantly reduced in presence of two Cdk4 inhibitors. These results confirm that these inhibitors render their neuroprotective ability by inhibiting the kinase activity of Cdk4. Next, we determined the effect of Cdk4 inhibitors on downstream effectors of apoptotic cell cycle pathway that are required for execution of neuron death. Bim is an important proapoptotic protein which is induced in neurons and plays a necessary role in neuron death following NGF deprivation or Ab treatment.