Fig. 5. Effects of LTD₄ on the activities of Raf-1, B-Raf and MEK in Int 407 cells. In (A) and (B), the cells were stimulated with 80 nM LTD₄ for the indicated periods of time, after which kinase assays were performed with anti-Raf-1 (A) and anti-B-Raf (B) immunoprecipitates (as described in the Materials and Methods). In (C), cells were transfected with an empty vector or a HA-tagged K^-Raf-1 expressing vector and then stimulated or not with 80 nM LTD₄ for 3 minutes. After 3 minutes, whole cell lysates were prepared and analyzed by immunoblotting with an anti-phospho-Erk-1/2, an anti-total-Erk-1/2 and finally an anti-HA antibody. In (D), cells were pre-incubated in the absence or presence of PTX (500 ng/ml for 2 hours), GF109203X (GFX; 30 µM for 30 minutes), TPA (1 µM for 24 hours; PKC deplet) and thereafter stimulated or not with 80 nM LTD₄ for 3 minutes. After 3 minutes, the Raf-1 kinase activities were performed as in (A). In (E), cells were pre-incubated in the absence or presence of PTX (500 ng/ml for 2 hours), PP1 (10 µM for 15 minutes) or PD98059 (50 µM for 30 minutes) and thereafter stimulated or not with 80 nM LTD₄ for 3 minutes. After 3 minutes, the MEK kinase activities were performed as in (B). Representative control blots of the different immunoprecipitates are shown in panels (A,B,D,E). The kinase activity values are expressed as a percentage of untreated control cells and given as mean±s.e.m. of four separate experiments. Statistically significant effects (compared with untreated cells) were evaluated using an unpaired Student's t-test, ^*P< 0.05 and ^**P< 0.01.

Return to article