Effect of ALK-1 signaling on hemangioblast development. iALK1 ES cells were differentiated into EBs. Dox was added to the culture medium from day 2 of EB differentiation. Cells were characterized at day 3.25 as follows: (A) Western blot for HA confirms induction of CA-ALK-1 in iALK-1 ES cells. (B) Western blot for Smads. Induction of CA-ALK-1 leads to increased levels of phosphorylated Smad1/5/8 (pSmad1/5/8), downstream targets of ALK1. Actin and GAPDH were used as loading control. (C) Quantification of phosphorylated Smad1. After normalization to GAPDH levels, results were plotted as ratio between phosphorylated Smad1/5/8 and total Smad1. (D) iALK-1 ES cells were assayed for hemangioblast activity in BL-MCM, which consists of methylcellulose containing VEGF, SCF, and TPO. Error bars indicate SE from 3 independent experiments performed in duplicate. (E) Gene expression analyses for Flk-1, Gata1, Gata2, Lmo2, and SCL. Error bars indicate SE from 2 independent experiments performed in duplicates. *P < .05, **P < .01