FDM cells express negligible levels of p53 after IL-3 withdrawal, but p53 reporter activity is slightly elevated. (A) Cells of the indicated genotypes were either treated with doxorubicin (in the presence of IL-3) or deprived of IL-3. Whole cell lysates were prepared, and proteins were resolved on SDS-PAGE. Western blots were probed with antibodies to p53 and β-actin (loading control). (B) Cells of the indicated genotypes were lentivirally transduced with a construct encoding a p53 transcriptional response element from the Puma promoter linked to GFP. p53 Transcriptional activity (GFP fluorescence intensity) was measured 24 hours after γ-irradiation or (C) 6 and 24 hours after cytokine withdrawal. The results shown are means ± SEMs of3 independent experiments, and n refers to the number of independent clones in each experiment. Asterisks denote a P value of .02, comparing Bax^−/−;Bak^−/− GFP expression before (time 0) and 24 hours after IL-3 withdrawal with the use of a t test (1-tailed, paired analysis).(D) Representative FACS plots of panels B and C are shown.