Since the 1990s, pRb was known to be inactivated via phosphorylation. Until, the prevailing model was that Cyclin D- Cdk 4/6 progressively phosphorylated it from its unphosphorylated to its hyperphosphorylated state (14+ phosphorylations). However, it was recently shown that pRb only exists in three states: un-phosphorylated, mono-phosphorylated, and hyper-phosphorylated. Each has a unique cellular function.
Before the development of 2D IEF, only hyper-phosphorylated pRb was distinguishable from all other forms, i.e. un-phosphorylated pRb resembled mono-phosphorylated pRb on immunoblots. As pRb was either in its active “hypo-phosphorylated” state or inactive “hyperphosphorylated” state. However, with 2D IEF, it is now known that pRb is un-phosphorylated in G0 cells and mono-phosphorylated in early G1 cells, prior to hyper-phosphorylation after the restriction point in late G1.