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Fig. 6 C-terminal region of RAM domain mediates the hnRNP I-induced NICD turnover.
(A) Schematic representation of serial deletion NICD constructs (left panel) and the Western blot results (right panel) showing that the RAM domain is required for hnRNP I-induced NICD turnover. Myc-GFP (pointed by arrow) was used as the control for microinjection and loading. * indicates NICD and NICD deletion constructs. (B) Schematic representation of RAM deletion constructs (left panel) and the Western blot results (right panel) demonstrating that the C-terminal region of RAM domain is required for hnRNP I-induced NICD turnover. Myc-GFP was used as the control for injection and loading (pointed by arrow). * indicates RAM and RAM deletion constructs. (C) Western blot result showing that overexpression of hnRNP I destabilized GST-RAMC protein purified from bacteria. GST-RAMC protein (250 pg per embryo) was injected alone, or together with hnRNP I RNA (2 ng) into Xenopus embryos. Control and injected embryos were harvested at the tailbud stage. A total volume of 1 ml lysate, which was prepared from 50 embryos, was incubated with 50 ml glutathione-agarose beads at room temperature for 1 hour to recover GST-RAMC protein. Protein was eluted from the beads and blotted with an anti-GST antibody (upper panel). Supernatants were probed with the anti-tubulin antibody (lower panel).