Transient expression of Bcl-2 constructs with defined subcellular localization in Jurkat cells. (A) Schematic representation of human wildtype Bcl-2 (Bcl-2 WT) localizing to both mitochondria and the ER, Bcl-2 ActA targeted to mitochondria, Bcl-2 cb5 expressed at the ER, and Bcl-2 ΔTM localized in the cytosol. The Bcl-2 homology domains BH1-BH4 are indicated together with the transmembrane domain (TM) for wildtype Bcl-2, which is replaced by amino acids from Listeria monocytogenes ActA, from rat cytochrome b5 (cb5) or deleted altogether in the other constructs. (B) amino acid sequence of the carboxyterminus of wildtype Bcl-2, Bcl-2 ActA, Bcl-2 cb5 and Bcl-2 ΔTM. The amino acids derived from ActA and cb5 are shown in bold. (C) Wildtype Jurkat cells were transiently nucleofected with empty vector pRc/CMV or with pRc/CMV encoding wildtype Bcl-2. 24 h after transfection, the cells were stimulated with 100 ng/ml hTNF in combination with 5 μg/ml CHX and 50 μM zVAD-fmk or left untreated for another 24 h. Prior to stimulation, the cells were preincubated for 60 min with 50 μM zVAD-fmk. PI-uptake was determined by flow cytometry and the percentage of viable cells is indicated in the lower right quadrants of the dot plots. One representative experiment out of three performed is shown. (D) Quantification of cell viability data. The bar graphs represent the means from all three independent experiments, error bars indicate the respective standard deviations. Due to the transfection procedure, the cells generally display a lower viability than untransfected cells (Fig. 1). (E) In parallel, expression of Bcl-2 in the transfectants was visualized by Western blot analysis. The band in vector transfectants represents endogenous Bcl-2.