FIGURE 7.
Caspase-3 interacts with caspase-2 that is influenced by p38 MAPK. A, BiFC experiment using split Venus constructs was done as described under “Experimental Procedures.” The amino-terminal (VN) half of Venus linked to pro-caspase-2 and the carboxyl-terminal (VC) half-linked to pro-caspase-3 were transfected for 24 h into Huh7 cells. To avoid an increase in cell death, we used the catalytically inactive mutant caspase-3-C163A for the expression. Huh7 cells were analyzed under a fluorescent microscope, and representative images are shown. Green fluorescence dots reveal the complementation of VN- and VC-halves of Venus showing an interaction between caspase-2 and caspase-3 in living cells. Size bar, 5 μm. B, cells were left untreated or stimulated with 5 ng/ml pro-NGF for 16 h. IP was done as described under “Experimental Procedures” using anti-caspase-2 antibodies followed by immunoblotting. Left panel, input. Middle panel, IP. Right, quantification of the relative degree of interaction. Pro-caspase-3 interacted with endogenous caspase-2 in control cells, but this was reduced in pro-NGF-treated cells. Values are mean ± S.E., n = 4. **, p < 0.01 for pro-NGF versus control. C, expression of DN-p38 MAPK inhibited the pro-NGF mediated decrease in the caspase-2-caspase-3 interactions. IP was done as above. Typical experiment is shown and was repeated. D, control experiments. There was no caspase immunoprecipitated using control IgG.
