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Fig. 1. Rsk is activated downstream of Mos-MAPK pathway in starfish oocytes.
(A) Specificity of antibodies against the N-terminal kinase domain of
starfish Rsk. Lysates from immature oocytes were separated on a 10% SDS-PAGE
gel and blots were probed with pre-immune sera and anti-Rsk sera (1 and 2),
respectively. Right side, molecular mass markers in kDa. (B) Dynamics
of Rsk during starfish meiotic cycles. Extracts were prepared from oocytes and
fertilized eggs at 10 minutes intervals after 1-MeAde addition, and
immunoblotted with anti-MAPK antibody (second panel) and anti-Rsk serum 2
(third panel). Immunoprecipitates with the anti-Rsk serum 1 were assayed for
phosphorylation of GST-S6. Rsk activity was detected on an autoradiogram
(fourth panel), which was then quantified (fifth panel). Extracts were also
assayed for phosphorylation of histone H1 (first panel). U, M and L indicate
three forms of Rsk. The upper and lower bands of MAPK correspond to the active
and inactive forms, respectively. Arrows and arrowheads indicate the time of
GVBD and fertilization, respectively. (C) Activation of MAPK and Rsk by
GST-Mos injection. Immature oocytes were injected with 25 pg of control GST
(left) or GST-Mos (right), recovered at 60 minutes, and immunoblotted with
anti-Rsk serum 2 (upper) and anti-MAPK antibody (lower). (D) Rsk
inactivation by MAPK inactivation. Maturing oocytes 50 minutes after 1-MeAde
addition were treated with 10 µM U0126, an inhibitor of MAPK kinase, or
control DMSO, and then recovered at 10-minutes interval. The extracts were
immunoblotted with anti-Rsk serum 2 (upper) and anti-MAPK antibody
(lower).
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