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Figure 3. Flowchart of the experimental procedure of the screen.A X. tropicalis library of unique, full-length clones has been established based on sequence comparison and clustering of over 1,220,000 ESTs, and rearrayed in a 96-well plate format. Pools of 8 mRNAs were prepared from pooled bacteria culture and in vitro transcription. Then in vitro transcribed mRNA pools were injected into fertilized X. laevis embryos at 1–2 cell stage. After microinjection, injected embryos were collected at stage 8 (blastula), stage 10.5 (gastrula), and stage 14 (neurula). Protein extracts from embryos were loaded onto SDS-PAGE for subsequent Western blot analysis. Antibodies used include anti-phospho-Smad1/5/8, anti-phospho-Smad2, anti-phospho-Akt, and anti-phospho-Erk. Once a potential active pool was identified, the pool was de-convoluted and single molecule injection was performed to identify the active molecule. Image published in: Zhang S et al. (2013) Image reproduced on Xenbase with permission of the publisher and the copyright holder. This image is reproduced with permission of the journal 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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