Figure 4. Effect of PNUTS knockdown versus Roscovitine treatment on Rb phosphorylation state.
p53−/− HCT116 cells were treated with DMSO or Rosc for eight hours or with nontargeting (NT) or PNUTS RNAi for twenty four hours followed by immunoblotting as described in the Materials and Methods. The antibodies utilized are indicated to the right of the figure. The antibodies to Rb amino acids 807/811, 780, 821, and 795 recognize the phosphorylated sites of Rb. However, the antibody to Rb-608 recognizes the unphosphorylated form of 608. Cleaved Parp is a marker of apoptosis (19). Total Rb expression is shown and PNUTS knockdown and equal loading were confirmed by examining expression of PNUTS and β-actin, respectively. Data shown is representative of two separate experiments.