Phosphorylation and dephosphorylation of the cdk provides additional control. DNA damage is a catastrophic event that delays progression in the cell cycle. Delay provides time for DNA repair or initiating programmed cell death. The real danger in mutating the DNA of a cell is not that the cell will die but that the cell may acquire uncontrolled proliferative capacity. As such, it could become cancerous.
Contact inhibition is an alternative to serum deprivation.
Assay for H1 kinase activity. This is a convenient substrate, but the biological signficance of this phosphorylation is uncertain. Rb is analyzed because it appears to be a bona fide substrate of G1 and S phase cyclins. Rb is classified as a tumor suppressor. In the dephosphorylated state, it binds and inhibits transcription factors such as E2F. The cdk-cyclin complex is thought to phosphorylate Rb causing Rb to dissociate from E2F. E2F will then be free to activate transcription of genes involved in DNA replication.