Presence of IL3 or SCF during the adhesion assay.

Presence of IL3 or SCF during the adhesion assay prevents up-regulation of p27^KIP1 and inhibition of cdk2-kinase. Mobilized blood CD34⁺ cells cultured for 48 hours in serum-free medium with low-dose cytokines were incubated with 8A2 and plated, with or without 10 ng/mL IL3, in PLL- or FN-coated dishes for 12 to 16 hours. Adherent and nonadherent cells were collected separately, and cells were lysed. Methods used to demonstrate presence and activity of p27^KIP1, cyclin-E, and cdk2-kinase activity are as described in legend to Figure 5. Differences in protein levels were evaluated by scanning images with a GS-700 Imaging Densitometer and quantitated with the use of Molecular Analyst software. A representative example of 2 individual experiments is shown. Relative protein levels/kinase activity values are shown below all lanes (PLL-nonadherent is arbitrarily 1).