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Fig. 6 Stability of WT- or M4A-Smad1 proteins in animal caps. (A) Embryos were injected with 1 ng mRNA encoding myc-tagged WTor M4A-Smad1. Animal caps were isolated at late blastula and collected for immunoblots at intervals between late gastrula (st. 12.5) and early tailbud stages. The myc-tagged proteins are easily detected, indicating that both proteins are translated well in vivo. The WT-Smad1 protein, however, accumulates at earlier stages, and persists at high levels longer than does the M4A-Smad1 protein. This effect was consistent across all experiments and multiple preparations of synthetic mRNA (n56 experiments). (B) Linearity of immunoblot detection. Embryos were injected with 1 ng mRNA encoding myc-tagged WT-Smad1 mRNA. Animal caps were isolated at the late blastula stage and lysed when controls reached st. 10.5. A 2-fold serial dilution was prepared from the lysate, and the dilution series was processed for immunoblotting. Band intensities were quantified and compared with predicted values. A representative experiment and quantification are shown. Image published in: Sater AK 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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