TAT-Pim1(wt) enhances survival in a PI3-kinase–independent manner. Basophils were preincubated with the buffer (control), or with TAT-Pim1(wt) and TAT-Pim1(KD) for 1 hour, and then wortmannin (100 nM) or LY2940002 (30 μM) was added to cell cultures for 24 hours as indicated. (A) Cell apoptosis was determined by annexin V–FITC staining and subsequent flow cytometry. Apoptotic cells are shown as percentage of annexin V–positive cells. Data are presented as mean plus SEM from 3 experiments. *P < .05, **P < .01, for the comparison of TAT-Pim1 versus medium control within the groups, by one-way ANOVA. (B) Activation of caspase 3 was assessed by immunoblotting measuring procaspase 3 proteolytic cleavage. TAT-Pim1 fusion proteins were detected using anti-HA antibody. β-Actin was used as loading control. The level of the activated caspase 3 was estimated in each sample by densitometry. Fold change of caspase 3 activity was calculated as described in the legend to Figure 4. (C) Phospho- and total of p38 MAPK were detected using specific antibodies. Fold change of p38 MAPK phosphorylation was calculated as relative ratio of the band intensity value of phosphorylated p38 MAPK in individual sample divided by the band intensity value of phosphorylated p38 MAPK from control basophils incubated in medium alone. The band intensity value of phosphorylated p38 MAPK was normalized to the band intensity value of total p38.