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Figure 4


Fig. 4. Inhibition of GSK-3beta blocks ceramide-induced activation of caspase-2 and caspase-8 and expression of tBid. 10I cells were treated with 25 µM C2-ceramide for 6 hours with or without LiCl (10 and 20 mM) and SB216763 (10 and 20 µM), or were pre-transfected for 24 hours with control siRNA (100 nM) or GSK-3beta siRNA (50 and 100 nM). (A) We used enzymatic cleavage of the specific substrates benzyloxycarbonyl-Val-Asp(-OMe)-Val-Ala-Asp(-OMe)-pNA and benzyloxycarbonyl-Ile-Glu(-OMe)-Thr-Asp(-OMe)-pNA to determine the activities of caspase-2 and caspase-8. OD, optical density. Data are given as the average of triplicate cultures (mean ± s.d.). (B) We used western blotting with a tBid-specific antibody to detect tBid expression. beta-actin levels were determined as an internal control.





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