Fig. 3

Inhibition of the MEK/Erk/Rsk pathway prolongs Ras activation. a Resting HeLa or MEF cells were left untreated or treated with the MEK inhibitor U0126 (10 μM), followed by EGF stimulation and analysis of Ras and Erk activity. b Same experiment as in A performed in H-Ras^-/-, N-Ras^-/-, K-Ras^lox/lox MEFs expressing only K-Ras. c HeLa cells pretreated with the MEK inhibitor U0126 or the PI3K inhibitor Wortmannin (30 min,100 nM) were challenged with EGF and subjected to a biochemical Ras activation assay. d HeLa cells expressing the trivalent affinity probe for Ras-GTP E3-R3(A/D) (see experimental section and Ref.[51]) were treated with U0126 or left untreated prior to stimulation with 10 ng/ml EGF. The time-dependent re-distribution of E3-R3(A/D) was imaged alive by confocal laser scanning microscopy. Probe relocation to the plasma membrane (marked by arrowheads) illustrates Ras activation. Over 30 cells monitored in 3-5 individual experiments responded with the same redistribution kinetics