Functional Analysis of CDK Inhibitor p21WAF1

p21^WAF1 was originally identified as a protein that binds and inhibits cyclin-dependent kinases (CDKs). p21^WAF1 is recognized to have at least two separate roles—first as a CDK inhibitor, and second as an inhibitor of PCNA, an accessory protein of DNA polymerase δ. p21^WAF1 plays a critical role in the cellular response to DNA damage. Additionally, p21^WAF1 plays a role in DNA repair, apoptosis, cellular senescence, terminal differentiation, and cell cycle arrest upon extracellular signaling. p21^WAF1 protein levels are regulated both by transcriptional control by p53 and by factors other than p53, as well as by posttranscriptional regulation. Although the role of p21^WAF1 has been explained so far only by its interaction with CDKs and with PCNA, it has several other binding partners. The ability of p21^WAF1 to participate in several cellular functions has been widely studied by transfection of cells with p21^WAF1 vectors. We describe here procedures for analysis of p21^WAF1 function in mammalian cells after transfection of p21 plasmids. The procedures include inhibition of DNA synthesis, cellular localization, association with binding partners, and half-life measurements.