Fig. 3. LH causes MAP kinase-dependent phosphorylation of multiple serines of Cx43. (A-D) Immunoblots of follicles, ±LH for various times, probed for total Cx43, and for phosphorylation on particular serines as indicated. The fastest migrating species, marked P0, contains the unphosphorylated form, and P1, P2 and P3 are three different commonly observed phosphorylated forms. (E) Densitometric analysis of the relative amount of phosphorylation on S262 and S279/S282 of Cx43, as a function of time after LH addition. The results are expressed as the ratio of the density of the phosphorylated bands divided by the density of the bands representing total Cx43 from the same blot, and are normalized to the maximum value obtained (at 0.5 or 1 hour) for the time series. The results of four independent experiments for each antibody were combined (mean±s.e.m.). A similar time course was seen in three independent experiments with the pS255 antibody, but the signal was too low to allow meaningful quantitation. (F) Immunofluorescence images of pS279/S282 Cx43 in antral follicles with or without a 1 hour exposure to LH. The images are representative of six follicles without LH, and 11 follicles with LH. (G) Immunoblots of follicles with or without a 1 hour exposure to LH, in the presence of 10 µm of the MEK inhibitor U0126 or its inactive analog U0124. The blots were probed for phosphorylation of MAP kinase and particular serines of Cx43, and also for vinculin (lower row), to confirm that protein amounts in each lane were equivalent. Similar results were obtained in three independent experiments.