Confirming the presence of negative feedback. HEK293 cells were pretreated with 5 μM U0126 or vehicle alone (DMSO) for 30 min prior to stimulation with 0.1 nM, 1 nM or 10 nM of EGF (A) or TGFα (B) for 5 or 30 minutes. Control cells were left unstimulated (−). Total cell lysates were assayed for activated RasGTP as described in “Materials and Methods” section. IP denotes the pull-down fraction, TCL denotes total cell lysate, and IB denotes immunoblot.