Involvement of exogenous CKIϵ in G-CSF–induced cell differentiation. (A) Expression of WT- or KN-CKI. Total cell lysates (TCLs) extracted from recombinant virus–infected 32D cell lines and immunoprecipitates (IPs) with anti-HA antibody were subjected to immunoblot analysis (IB) by using anti-CKIδ and CKIϵ antibodies. Black arrowheads indicate the position of endogenous 49-kDa CKIδ or 47-kDa CKIϵ. White arrowheads indicate HA-tagged recombinant CKIδ or CKIϵ. (B) Cell growth of virus-infected 32D cell lines in the presence of WEHI-CM (IL-3) or G-CSF. Each cell line was subcultured at 5 × 105 cells/well in a medium containing 15% WEHI-CM (IL-3) and in a differentiation medium with 10 ng/mL G-CSF on day 0. Viable cells after staining with trypan blue were counted at days 1, 2, 3, and 4. Values are the means of cell numbers/well ± SE of four samples in 5 independent experiments (*P < .05 vs control cells). (C) Cell differentiation induced by 10 ng/mL G-CSF was assessed daily by flow cytometry (FACScan; Becton Dickinson, San Jose, CA) by using Mac-1 antibody with phycoerythrin-conjugated streptavidin (Becton Dickinson) (upper panels: WT-CKIδ- and KN-CKIδ-32D cells; lower panels: WT-CKIϵ- and KN-CKIϵ-32D cells). Daily histogram analysis of Mac-1 expression is a representative 1 of 5 similar results. Data represent the mean ± SE of Mac-1 expression of 5 independent experiments (*P < .05 vs control cells).
