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FIG. 7. Expression of xARH dominant negative mutant (xARH-DM) in cultured oocytes inhibited vitellogenin endocytosis. A, schematic diagram of xARH-DM showing mutations in the clathrin box (LLDLE) and the 258 Dφ(F/W) motif. B, stage III oocytes were incubated in culture medium containing biotinylated VTG for various periods of time. Surface-bound VTG was removed by washing with a high pH buffer. The internalized biotinylated VTG was detected using streptavidin-HRP conjugate. The nitrocellulose membrane was stained with Ponceau S to visualize the lipovitellin band used as a loading control. Biotin-VTG was progressively internalized over the culture period. C and D, after the injection of 5 ng of xARH-DM or control galactosidase (Gal) transcripts, oocytes were cultured overnight. The expression of Myc-tagged xARH-DM protein was confirmed by Western blot analysis (C), and VTG uptake was measured (D) as described in B. xARH-DM severely inhibited VTG uptake compared with the galactosidase control. Image published in: Zhou Y et al. (2003) Copyright © 2003. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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