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Morpholino knock-down of Nipbls. (A) Total protein lysates (10 embryos per lane) from uninjected embryos or embryos injected with either 0.5 ng of nipbla-MO1 or 0.5 ng of nipblb-MO1 were prepared at 10 and 24 hpf and subjected to Western blotting with anti-Nipbla and Nipblb-antibodies. Positions of Nipbla and Nipblb proteins are shown on the right. A lower band recognized by anti-Nipbla antibody (*) at 24 hpf is not reproducibly observed and may be a degradation product of Nipbla. (B) Activities of nipblb-MOs at earlier stages. EGFP reporter RNAs (100 pg/embryo)—nipbla-5′-UTR-EGFP (upper panels) and nipblb-52-UTR-EGFP (lower panels)—were injected alone or together with either nipblb-MO1 or nipblb-MO2 (1 ng/embryo), and EGFP fluorescence measured at 10–11 hpf. Views are lateral, with anterior and dorsal to the top and right, respectively. (C) Morphology of living embryos at indicated stages. Uninjected embryos, control embryos co-injected with 0.75 ng each of two 5-mis-nipbl-MOs, and embryos injected with either nipbla-MO1 (0.75 ng) or nipblb-MO1 (0.75 ng) alone or together (nipbla/b-morphants) are shown. Pericardial edema and tail defects are indicated by asterisks and arrowheads, respectively. Views are lateral, with dorsal to the top. Scale bar: 100 μm.
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