Figure 5.

In CML CD34⁺ cells, >80% of p27^Kip is located in the cytoplasm and does not relocate to the nucleus after adhesion of cells to FN. Nuclear and cytoplasmic proteins were isolated separately from 10⁷ CD34⁺ cells either freshly selected (A), or recovered in the adherent and nonadherent fraction of adhesion assays (B). Proteins present in cytoplasm or nucleus were resolved by SDS/PAGE, and blots were probed with anti-p27^Kip antibodies and goat anti-mouse HRP-conjugated antibody. Blots were then stripped and reprobed with anti-cdk2 antibodies and goat anti-mouse HRP-conjugated antibody, and stripped again and probed with anti-cyclin-H antibodies and goat anti-mouse HRP-conjugated antibody. A representative example of three experiments is shown.