FIG. 4.

pRb does not affect the phosphorylation state of a prelabeled Py T Ag. Increasing concentrations of pRb were added to complete replication reactions containing ³²P-labeled Py T Ag. After 3 h of incubation at 33°C, the mixtures were split to two. (A) One half was analyzed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted by PAb F5 (Western) prior to autoradiography (Autorad). (B) From the other half (25 μl), DNA was purified from the replication mixtures, linearized, digested with DpnI, and examined on 1% agarose gel and autoradiography. Form I DNA is indicated on the right; sizes of markers are indicated in kilodaltons on the left.