Kinase-dead Pim1 inhibits IL-3–induced basophil survival. (A) Schematic diagram representing the structure of TAT-Pim1(wt) and TAT-Pim1(KD). TAT peptide precedes the HA-tag at the N-terminus of Pim1, 6 His residues are located at the C-terminus of fusion protein. Kinase dead mutant Pim1(KD) has a single amino acid substitution of Lys67 to Met (K67M). (B) Expressed in E coli BL21(DE3) and further purified TAT-Pim1(wt) and TAT-Pim1(KD) fusion proteins were analyzed by SDS-PAGE followed by Coomassie blue staining. (C,D) Basophils were treated with buffer (control) or transduced with TAT-Pim1(wt) or TAT-Pim1(KD) as indicated and cultured in the presence of IL-3 for 24 hours. Cells cultured in the absence of IL-3 are shown as controls. (C) Apoptosis was determined by annexin V–FITC staining and flow cytometry. Data are shown as mean plus SEM from 3 experiments. **P < .01 for comparison of IL-3 control versus TAT-Pim1(KD) + IL-3. (D) Activation of caspase 3 was assessed by immunoblotting for procaspase 3 and cleaved caspase 3. TAT-Pim1 fusion proteins were detected using anti-HA antibody and after the stripping with anti-Pim1 antibody. β-Actin was used as loading control. Fold change of caspase 3 activity was calculated as relative ratio of the band intensity value of cleaved caspase 3 in individual sample divided by the band intensity value of cleaved caspase 3 from control IL-3–treated basophils. The band intensity values of cleaved caspase 3 were normalized to β-actin.