Fig. 3. Effect of PD98059 or U0126 on phosphorylated MAPK (P-MAPK) and MPF activity in unfertilized eggs of P. lividus and L. pictus. (A) Comparison of ERK-LP in eggs of two sea urchin species using three different antibodies. (Aa) Samples 2, 3, 5 and 7 were cut in two halves after transfer and before blotting with various antibodies, and then re-aligned before ECL. The anti-ERK1 (ERK1) and the anti-panERK (panERK) antibodies detected in both species a doublet at approximately 44 kDa (small arrows) with an upper band corresponding to the protein detected with the anti-phosphoMAPK42/44 antibody (P-ERK). The anti-panERK antibody also revealed a single protein of approximately 81 kDa (large arrow). (Ab) Detection in P. lividus eggs of changes in the phosphorylation of the 44 kDa ERK-LP using the anti-phosphoMAPK42/44 antibody (left panel). A decrease in this signal was observed during the 1-hour treatment with 5 µM PD98059 but no change (small arrow) was observed after stripping and re-blotting with the anti-panERK antibody (right panel). A protein was detected by both antibodies (especially by the anti-pan ERK antibody) at approximately 81 kDa (large arrow). (B) Effect of 2.5 µM PD98059 on P-MAPK and MPF activity. (Ba) Fluctuations in the phosphorylation of ERK-LP were detected in P. lividus eggs with the anti-phosphoMAPK42/44 antibody (P-MAPK) and that of Cdc2-TyrP using the anti-Cdc2-TyrP antibody (upper panel). H1 kinase activity (lower panel) slowly increased after addition of the inhibitor, with transient decreases at times when Cdc2 was phosphorylated at tyrosine residue 15 (arrows). (Bb) Progressive loss of PD98059 activity after dilution in ASW. Changes after 1 µM PD98059 addition in P-ERK-LP after western blot using the anti-phosphoMAPK42/44 antibody. The inhibitor was added in the egg suspension at t=0. The supernatant, containing the inhibitor, was taken and added to fresh eggs at different times (arrow) that were then analyzed 30 minutes later (star). PD98059 was unable to act on the MAPK pathway after a 60-minute dilution in ASW. (C) Effect of 1 µM U0126. (Ca) L. pictus. U0126 addition induced oscillations in ERK-LP and in Cdc2-TyrP phosphorylation similar to those observed with PD98059 (Fig. 3B). No change was detected with the anti pan-ERK antibody after stripping and reprobing of the western blot performed with the anti-phosphoMAPK42/44 antibody. The same gel was cut around 38 kDa after transfer in order to blot the upper part for P-MAPK and the lower part for Cdc2-TyrP detection. (Cb) P. lividus. The upper panel shows that ERK-LP was dephosphorylated after addition of U0126, but was not phosphorylated again. Oscillations in Cdc2-TyrP similar to those observed in (Ca, upper panel) or after PD98059 (Fig. 3B) corresponded to a gradual increase in H1 kinase activity (lower panel) that showed decreases at times when Cdc2-TyrP level was high (arrows). (Cc) Progressive loss of U0126 activity after dilution in ASW. Changes after 0.5 µM U0126 addition in P-ERK-LP after western blot using the anti-phosphoMAPK42/44 antibody. The inhibitor was added in the egg suspension at t=0. The supernatant containing the inhibitor was taken and added to fresh eggs at different times (arrow) that were analyzed 30 minutes later (^*). A complete loss of U0126 activity was observed after a 3-hour dilution in ASW.