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FIGURE 4. Immunoblot and RT-PCR analyses demonstrating GDF5 requires both Furin and SPC6 to produce mature protein and become biologically active. A, immunoblot analysis of secreted proteins following mRNA injection of Xenopus oocytes. Xenopus oocytes (stage VI) were isolated, defolliculated, and injected with mRNAs encoding GDF5-T7 (25 ng), GDF5-T7 (25 ng) + Furin (25 ng), GDF5-T7 (25 ng) + SPC6 (25 ng), or GDF5-T7 (25 ng) + Furin (12.5 ng) + SPC6 (12.5 ng). Injected oocytes were incubated at 18 °C for 24 h, and oocyte supernatants were prepared for analysis as described under “Materials and Methods.” Arrows indicate the locations of the pro- and mature forms of GDF5-T7. Similar results were obtained in three separate experiments. B, conjugated animal caps (as indicated), produced at stage 9 and cultured to stage 17, analyzed by RT-PCR for the mesodermal marker Brachyury and the ventral markers Xhox3, and Xvent-1. Histone H4 is included as a loading control. Xenopus embryos were injected equatorially at the four-cell stage with mRNAs for either GFP (500 pg), Furin (150 pg) + SPC6 (150 pg) + GFP (200 pg), GDF5 (100 pg) + GFP (300 pg), GDF5 (100 pg) + Furin (150 pg) + SPC6 (150 pg) or GDF5 Mut Lys → Arg (100 pg) + GFP (300 pg). Image published in: Thomas JT et al. (2006) Copyright © 2006. 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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