Figure 4.

The effect of PCNA on p21 C inhibition of NER. (A) Ethidium bromide gel (top) and phosphorimage of dried ethidium bromide gel (bottom). Increasing amounts of PCNA (lane 1, molecular weight marker; lane 2, control; lane 3, 0 μM PCNA; lane 4, 0.2 μM PCNA; lane 5, 1 μM PCNA) were incubated with nuclear extract containing p21 C at 2 μM for 10 min at 30°C. Reaction buffers, UV-damaged plasmid DNA, and undamaged plasmid DNA were then added. The reactions were incubated at 30°C for 1 h. The samples were then subjected to RNAse A and proteinase K digestion. After extraction with organic solvents and ethanol precipitation, the plasmid DNA samples were linearized with EcoRI and resolved on a 0.75% agarose gel. The agarose gel was dried under heated vacuum, and the bands containing radioactively labeled DNA were quantified on a PhosphorImager (Molecular Dynamics). (B) Histogram of the results shown in A.