Induction of C/EBPα and PU.1 by G-CSF is inhibited in 32D cells expressing FLT3/ITD. The 32D/pBabePuro, 32D/FLT3, and 32D/FLT3/ITD cells were washed and transferred from medium containing IL-3 (1 ng/mL) to medium containing G-CSF (20 ng/mL) for 9 days. Every other day, fresh G-CSF–containing medium was added to cells to replace the old medium. (A) Total cellular RNA from 32D/pBabe (lanes 1-6), 32D/FLT3 (lanes 7-12), and 32D/FLT3/ITD (lanes 13-18) cells was prepared from 1 × 10⁷ cells after 0, 1, 3, 5, 7, and 9 days in G-CSF. RNA (10 μg) from each time point was then subjected to Northern blotting with a 1.1-kb NcoI-NcoI fragment of rat C/EBPα (top). The same membrane was stripped and then reprobed with actin cDNA probe (bottom). (B) Total protein lysates from 32D/pBabe (lanes 1-5), 32D/FLT3 (lanes 6-10), and 32D/FLT3/ITD (lanes 11-15) cells were prepared from 1 × 10⁷ cells after 0, 1, 2, 3, and 4 days in G-CSF. Protein lysates (50 μg) from each time point were then subjected to Western blotting with antibody against PU.1 (top). The same membrane was stripped and then incubated with antibody against HSP 90 (bottom).