CEP-701 restores G-CSF–mediated induction of C/EBPα and PU.1 expression in 32D/FLT3/ITD cells. (A) 32D/FLT3/ITD cells were treated without (lanes 1-7) or with (lanes 8-14) CEP-701 (5 nM) in the presence of G-CSF (20 ng/mL) with replacement of the medium every other day. Total cellular RNA was prepared from 1 × 10⁷ 32D/FLT3/ITD cells on days 0, 1, 3, 5, 7, 9, and 11. RNA (10 μg from each sample) was then subjected to Northern blotting with C/EBPα and actin cDNA probes. (B) Total protein lysates were prepared from 1 × 10⁶ 32D/FLT3/ITD cells on days 0, 1, 3, 7, and 9 treated without (lanes 1-5) or with (lanes 6-10) 5 nM CEP-701 in the presence of G-CSF. The whole protein lysates from each time point were then subjected to Western blotting with antibody against C/EBPα and HSP 90. Protein lysates from parental 32D cells (lane 11) were used as a positive control. (C) Total protein lysates were prepared from 1 × 10⁷ 32D/FLT3/ITD cells on days 0, 1, 2, 3, 4, and 5 treated without (lanes 1-6) or with (lane 7-12) 5 nM CEP-701 in the presence of G-CSF. Protein lysates (50 μg) from each time point were then subjected to Western blotting with antibody against PU.1 and HSP 90.