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Fig 1. Dnd1 is necessary and sufficient for nanos1 translation. (A) nanos1 (4 or 5 ng) was injected into oocytes with or without RNAs encoding Dnd1, Vasa, DeadSouth, Centroid and Dazl, each at 2 ng. Nanos1 protein was immunoprecipitated and analyzed by western blot. nanos1δTCE served as a positive control for nanos1 translation. The size difference between Nanos1δTCE and the wild-type Nanos1 is due to the deletion of the TCE, which is located immediately downstream of the translation initiation site. Experiments were repeated five times. (B) Quantification of band intensity of the western blot shown in A using ImageJ. (C) nanos1 RNA was added to wheat germ extracts with or without purified Dnd1 protein. Samples were analyzed for Nanos1 protein expression by western blot. nanos1δTCE served as a positive control. Experiments were repeated twice. (D) qPCR shows the levels of dnd1 and nanos1 in control and dnd1-depleted embryos (AS-oligo) at the 8-cell stage. Data are shown as mean±s.d. **P<0.01. Experiments were repeated three times. (E) Representative IF images show attenuation of endogenous Nanos1 protein expression by antisense depletion of maternal dnd1 (AS-oligo). Embryos were co-stained for Xiwi (green) and Nanos1 (red) at the 8- to 16-cell stage. Experiments were repeated twice. |
