Fig. 6. LTD₄-induced activation of Ras and its role in Erk-1/2 activation. In (A), cells were stimulated with 80 nM LTD₄ for the indicated periods of time or with 100 ng/ml EGF for 5 minutes, after which the cells were lysed. The active form of Ras was then precipitated with the minimal RBD fragment of Raf-1 fused with GST and then separated by SDS-PAGE and immunoblotted with an anti-Ras antibody. A similar analysis is shown in the top panel of (B), but here the cells were pre-incubated in the absence or presence of the Ras inhibitor FTI-277 (20 µM for 48 hours) or transfected with HA tagged N17 Ras as indicated and then stimulated with LTD₄ (80 nM) for 1 minute or EGF (100 ng/ml) for 5 minutes. In the second panel of (B), whole lysates from the samples used in the top panel were separated by SDS-PAGE and immunoblotted with an anti-Ras antibody. The blot show a gelshift of Ras derived from FTI-277 treated cells and the two bands of Ras from cells transfected with HA-tagged N17 Ras (the lower endogenous Ras band and the upper HA-tagged Ras band). In the third and fourth panels, the blot was probed with an anti-phospho-Erk-1/2 antibody and then reprobed with an anti-total-Erk-1/2 antibody. The relative densities given in the figure refer to densitometric analysis of LTD₄ and EGF-induced activation of Ras and Erk-1/2. The blots shown are representative of four separate experiments.