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Fig. 3. U0126 inhibits nuclear accumulation of GFP-PCNA, activation of ERK1, and
DNA synthesis in early embryos (A-F) and also in ionophore-activated eggs
(G-H). (A) Nuclear accumulation of GFP-PCNA and subsequently NEB are prevented
by 100 µM U0126, but not by 20 µM U0126 or 100 µM U0124 in early
embryos. Times after fertilization are indicated. Scale bar: 50 µm. (B)
Intensity of GFP-PCNA nuclear fluorescence in early embryos treated as in A
and, in addition, in unfertilized eggs incubated in 20 µM and 100 µM
U0126. Each graph represents the mean fluorescence value from three to five
different experiments; in each experiment different females were used. (C) DNA
synthesis is prevented by 100 µM U0126 in embryos. Incorporation of
[3H]-thymidine in embryos (control, white circle; treated with
U0126, black circle). (D) U0126 alone does not trigger DNA replication in
unfertilized eggs (black circle), but treatment with the Ca2+
ionophore A23187 (20 µM) as a control does induce DNA synthesis (white
circle). (E) U0126 (100 µM) applied 10 minutes before fertilization
abolishes the post-fertilization peak in ERK1 activity. We measured MAP kinase
activity in cell extracts during the first 8 minutes after fertilization:
control, black circle; after U0126 treatment, black square. (F) Western
blotting of immunoprecipitated active MAP kinase dimers, detected with
anti-dual phosphorylated MAP kinase antibody (New England Biolabs). Control, 0
and 6 minutes; 100 µM U0126 treated (+U0126), 0 and 6 minutes; control
immunoprecipitation in the absence of antibody (-AB, at 6 minutes). (G)
Wide-field fluorescence images and intensity of nuclear fluorescence of
unfertilized eggs microinjected with GFP-PCNA, incubated in 100 µM U0126
and activated with Ca2+ ionophore (A23187, 20 µM), together with
controls. Nuclear fluorescence was not observed in eggs treated with U0126.
Scale bar: 50 µm. (H) Incorporation of [3H]-thymidine in
unfertilized eggs treated with 20 µM Ca2+ ionophore A23187.
Ionophore activated eggs (white circle); eggs treated with U0126 15 minutes
before ionophore activation (black circle).
