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2+ indicator dye Fura 2 (Molecular Probe) (20 μM) and m1AChR mRNA (13 ng per embryo) were micro-injected at the 2-cell stage, and then mAb (40 ng per embryo) was injected at the animal poles. Animal pole blastomeres were cut off between the 32-to 128-cell stage. Changes in the fluorescence intensities excited at 340 and 380 nm upon carbachol addition (100 μM) were recorded and analyzed.
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We thank M. Asashima and M. Yuge for invaluable advice. N. Ueno for the Xenopus BMP4 construct, S. Numa for the m1AchR construct, Y. Nishiuchi for synthesis of peptide, Y. Eto for purified human recombinant activin A. K. Okabe, Y. Takeyama, and H. Manki for technical assistance, T. Natsume for discussions, and N. Ueno, J. Aruga, and M. Ohara for critical comments on the manuscript. Supported by grants from the Ministry of Education, Science, Sports and Culture of Japan (H.O. and K.M.) and grants from research fellowships of the Japan Society for the Promotion of Science for Young Scientists (S.K.).
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