Fig. 4. Identification of possible PKC isoform(s) involved in LTD₄-induced Erk-1/2 activation. In (A), cells were pre-incubated in the absence or presence of PTX (500 ng/ml for 2 hours) and then stimulated with or without 80 nM LTD₄ for the indicated periods of time. Membrane and cytosolic fractions were isolated, separated on SDS-PAGE and immunoblotted with the indicated specific anti-PKC isoform antibodies as described in the Materials and Methods. In (B), cells were pre-incubated in the absence or presence of 10 µM MAPTAM for 1 hour or 1 µM ionomycin for 5 minutes and thereafter stimulated or not with 80 nM LTD₄ for 3 minutes after which they were lysed. The cell lysates were then separated by SDS-PAGE and immunoblotted with an anti-phospho-Erk-1/2 antibody and then reprobed with an anti-total-Erk-1/2 antibody. In (C), cells were pre-incubated in the absence or presence of rottlerin (10 or 30 µM for 30 minutes), stimulated or not with 80 nM LTD₄ for 3 minutes and then lysed. The cell lysates were separated by SDS-PAGE and immunoblotted as in (B). In (D), the results in the left panel were obtained from cells treated with 1 µM TPA for the indicated periods of time after which whole-cell lysates were analyzed by immunoblotting for PKC isoforms , and . The right panel of (D) show results from cells pre-incubated with TPA, as in the left panel, but then stimulated or not with 80 nM LTD₄ for 3 minutes. After these 3 minutes, whole-cell lysates were prepared and analyzed by immunoblotting with an anti-phospho-Erk-1/2 antibody and then reprobed with an anti-total-Erk-1/2 antibody. (E) shows the results from cells transfected with either an empty vector (lanes 1-2), a RD-PKC-expressing or a RD-PKC-expressing vector that were 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 antibody and then reprobed with an anti-total-Erk-1/2 antibody and finally an anti-GFP antibody. (F) shows results from cells transfected with an empty vector (lanes 1-2) or a vector expressing K^-PKC that were stimulated or not with 80 nM LTD₄ for 3 minutes. After 3 minutes, whole-cell lysates were prepared and analyzed by immunoblotting for phospho-Erk-1/2, total Erk-1/2 and finally K^-PKC. All blots in this figure are representative of at least three separate experiments.