Figure 5.

Antibodies against each pRB family member immunoprecipitate their chromatin-bound targets. (A) Schematic representing the position of chromatin in gradients relative to free DNA and protein under conditions in which cells were either fixed with formaldehyde (+ cross-linker) or left untreated (− cross-linker). Brackets indicate chromatin fractions selected for further analysis in B. (B) Fractions in A were immunoprecipitated with anti-pRB, anti-p107, and p130 antibodies as indicated and the resulting precipitates were probed with the same antibodies as indicated. (C) p107 and p130 are detected at similar levels in T98G cells entering a second cell cycle (32-hr time point in Fig. 2). Chromatin immunoprecipitations were performed with the indicated antibodies, and E2F-1, cyclin A, and actin promoter fragments were amplified as described in the legend to Fig. 1. Distinct monoclonal and polyclonal anti-pRB antibodies were tested as shown.