ALK-1 rescues defective hemangioblast and primitive erythroid development in Eng^−/− ES cells. (A) Flow cytometry analysis of inducible iALK-1:Eng^−/− ES cells. FACS analysis was performed to detect GFP expression, which indicates the induction of ALK-1. Green line represents levels of GFP in undifferentiated ES cells, as well as cells from EBs differentiated for 3.75, 4.75, or 6.25 days cultured in the presence of dox. The gray line denotes controls (no dox). (B) Western blot analyses of Smads at day 3.75 nonsorted EBs. 25μg proteins were subjected to SDS-PAGE and Western blotting. pSmad1/5/8 indicates phosphorylated Smad1/5/8. (C) Quantification of phosphorylated Smad1/5/8. After normalization to Gapdh levels, results were plotted as ratio between phosphorylated Smad1/5/8 and total Smad1. (D) iALK-1:Eng^−/− ES cells were assayed for BL-CFC and primitive erythroid development by plating 1 × 10⁵ cells from EBs differentiated for 3.75 and 4.75 days, respectively. Error bars indicate SE from 3 independent experiments performed in duplicate. (E) Gene expression analyses for embryonic globin. Transcripts are normalized to Gapdh. Error bars indicate standard errors from 3 independent experiments performed in duplicate. (F) Representative morphology of blast colonies obtained from Eng^−/− and rescued iALK-1:Eng^−/− ES cells. Colonies are shown at the same magnification (200×). **P < .01.