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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.
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