Adhesion to FN.

Adhesion to FN is associated with increased levels of p27^KIP1 and decreased cdk2-kinase activity. Mobilized blood CD34⁺ cells cultured for 48 hours in serum-free medium with low-dose cytokines were incubated with 8A2, washed, and plated in PLL- or FN-coated dishes for 12 to 16 hours. Adherent (Adh) and nonadherent (NA) cells were collected separately, and cells were lysed. A representative example of 3 individual experiments is shown. For p27^KIP1, protein extracts were separated by SDS-PAGE, transferred onto nitrocellulose, and incubated with antibodies against p27^KIP1 and goat antimouse HRP-conjugated antibody. Cyclin-E was immunoprecipitated from protein-G-agarose beads. Immune complexes were separated by SDS-PAGE and blots probed with anti–cyclin-E antibodies and goat antimouse HRP-conjugated antibody. Protein bands were visualized with the use of the ECL detection system, and cdk2 was immunoprecipitated with the use of protein-G-agarose beads. The immune complexes were separated by SDS-PAGE, and blots were probed with anti-cdk2 antibodies and goat antimouse HRP-conjugated antibody. Cdk2 activity was assayed by adding 5 μg histone and 10 μCi [r-³²P]. Reaction products were resolved by SDS-PAGE, and the gel was exposed to X-ray film. Differences in protein levels were evaluated by scanning images with a GS-700 Imaging Densitometer and quantitated with the use of Molecular Analyst software. Relative protein levels/kinase activity values are shown below all lanes (PLL-nonadherent is arbitrarily 1).