Fig. 2. Characterization of LTD₄-induced activation of Erk-1/2. Cells were incubated in the absence or presence of 80 nM LTD₄ and the indicated inhibitors. Cell lysates were separated by SDS-PAGE and immunoblotted with antibodies specific for phosphorylated Erk-1/2. Thereafter, the blots were reprobed for total Erk-1/2. (A) outlines a representative blot and the accumulated concentration curve of LTD₄-induced Erk-1/2 phosphorylation. Cells were treated with the indicated concentrations of LTD₄ for 3 minutes with or without ZM-198,615 (ZM; 50 µM, 15 minutes) after which their degrees of Erk-1/2 phosphorylation were determined. (B) shows a representative blot and graph of the accumulated time course of LTD₄-induced Erk-1/2 phosphorylation. Cells were treated with 80 nM LTD₄ for the indicated periods of time, after which Erk-1/2 activities were assayed as described in the Materials and Methods. (C) Cells were pre-incubated in the absence or presence of PTX (500 ng/ml for 2 hours), GF109203X (GFX; 30 µM for 30 minutes), PP1 (10 µM for 15 minutes) or PD98059 (50 µM for 30 minutes). The cells were then incubated in the absence or presence of 80 nM LTD₄ for 3 minutes after which their Erk-1/2 activities were assayed. In (D), cells were pre-incubated in the absence or presence of Rp-cAMPS (50 µM for 30 minutes), wortmannin (100 nM for 10 minutes) or LY294002 (50 µM for 30 minutes) and incubated with or without 80 nM LTD₄ for 3 minutes after which their Erk-1/2 activities were assayed. The blots shown are representative of at least five separate experiments. Values obtained from the densitometric analyses are calculated as a percentage of untreated control cells and given as mean±s.e.m. of five separate experiments.

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