Test compounds inhibit ERK-mediated phosphorylation of p90Rsk-1 and Bad. (A) HeLa cells were pre-treated for one hour in the presence or absence of 50 uM of the indicated test compounds and then stimulated with EGF (25 ng/ml) for 10 minutes. Immunoblots of phosphorylated p90Rsk-1 (pRsk), total Rsk (Rsk), phosphorylated ERK1/2 (ppERK), and total ERK2 (ERK2). α-tubulin was used as a loading control. Graph shows densitometry quantification of pRsk-1 to total Rsk or ppERK2 to ERK2 ratios. Data represents the mean ± SEM from three independent experiments. (B) In vitro kinase assays examining 32P incorporation into p90Rsk-1 following incubation with active ERK2 and γ-32P-ATP for 60 min. in the absence or presence of 1-25 μM of test compounds. Relative phosphate incorporation was quantified by phosphoimager analysis. (C) HeLa cells were serum starved overnight and pre-treated for 1 hr with 50 uM indicated test compounds, 10 mM U0126 (U), or 25 mM LY294002 (L) prior to stimulation with or without EGF (25 ng/ml). Immunoblot analysis of Bad phosphorylated on Ser112 (pS112) or Ser136 (pS136) and total Bad. α-tubulin was used as a loading control. Data represents the mean ± SEM from three independent experiments. * and # indicates statistical significance compared to EGF-only treatment (A and C) or untreated controls (B), p ≤ 0.05 and p ≤ 0.01, respectively. C, untreated control; (-) EGF treated control.