FIG. 5.

Lack of significant leakage of PCNA. To evaluate the leakiness of the nuclei during the in vitro assay, the presence in the flowthrough of spin columns of PCNA, a nuclear resident protein, was examined by Western blotting as described in Materials and Methods. The total PCNA nuclear pool is shown by loading the same number of nuclei as in the assay. The results indicate that both (A) WT and (B) V701 nuclei are relatively stable, with a minimal release of PCNA. Note that upon prolonged exposure, a signal can be seen for V701. The negative-control “Buffer” was the in vitro assay buffer.