Figure 3.

Expression of DD-DAPk protects from TNF-α– and Fas-induced cell death. (a) Expression of endogenous DAP-kinase in cell lines. Western blotting analysis of extracts of indicated cells, using anti–DAP-kinase antibodies. (b) Expression of recombinant DD-DAPk. Extracts from cells transfected with DD-DAPk construct were immunoprecipitated by anti-Flag antibodies and analyzed by Western blotting using anti-Flag. (c) Transient transfection of 293, HeLa, or MCF7 cells with vectors encoding p55-TNF-R, GFP, and either luciferase (Luc), death domain of DAP-kinase (DD-DAPk), or dominant negative mutant of FADD/MORT1 (DN-MORT). The percentage of apoptotic cells was calculated as described in Materials and Methods. (d) Same as c, except that p55/Fas chimera was used instead of p55-TNF-R. (e) Transient transfection of HeLa cells with vectors encoding GFP and either luciferase, DD-DAPk, or DN-MORT. Cells were treated 24 h after transfection with a combination of TNF-α and cycloheximide. The number of apoptotic cells was scored under fluorescent microscopy 3 h after treatment.