Figure 6

Test compounds inhibit ERK-mediated phosphorylation of caspase-9 in cells and in in vitro kinase assays. (A) HeLa cells transfected with caspase-9 (C287A) were pre-treated for 1 hour with 50 uM of indicated test compound, stimulated with EGF (25 ng/ml), and immunoblotted for phosphorylated caspase-9 (pT125) or total caspase-9. Graph shows the mean ± SEM for relative phosphorylation as determined by densitometry scanning from 3 independent experiments. (B) In vitro kinase assays examining ³²P incorporation into caspase-9 following incubation with active ERK2 and γ-³²P-ATP for 60 min. in the absence or presence of 1-25 μM of the indicated test compound. Relative phosphate incorporation was quantified by phosphoimager analysis. Data show the mean ± SEM of three independent experiments. * and # indicates statistical significance compared to EGF-only treatment (A) or untreated controls (B), p ≤ 0.05 and p ≤ 0.01, respectively. C, untreated control; (-), EGF treated control; U, U0126