FIG. 5.

Inhibition of PI 3-kinase prevents cytokine treatment from overriding DNA damage-induced inactivation of cyclin E-Cdk2 and expression of p27. (A) 32D-EpoR[wt] cells were synchronized in G₀/G1 in the absence of growth factor (No factor). Synchronized cell cultures were supplemented with LY294002 (10 μM) or vehicle control (dimethyl sulfoxide [DMSO]), plus Epo or IL-3. Parallel cultures were then left untreated or exposed to γ irradiation (+ 4 Gy). All cultures were incubated for an additional 24 h prior to analysis by flow cytometry. Values represent the percentage of cells in S phase. (B) Cell lysates prepared from the cultures represented in panel A were immunoprecipitated with Cdk4 or Cdk2 antibodies, and in vitro kinase assays were performed. Alternatively, total cell lysates were Western blotted with antibodies specific for Cdk2 or p27.