ALK-5 gain-of-function impairs hemangioblast development. (A) Western blot against HA confirming the induction of CA-ALK-5. (B) Western blot for Smads. Induction of ALK-5 leads to increased levels of phosphorylated Smad2 (pSmad2), downstream target of ALK-5. Actin and Gapdh were used as loading control. (C) Quantification of phosphorylated Smad2. After normalization to Gapdh levels, results were plotted as ratio between phosphorylated Smad2 and total Smad2. (D) iALK-5 ES cells were assayed for hemangioblast activity in BL-MCM on day 3.25 of EB differentiation. The graph represents 3 experiments performed in duplicate. Bars indicate SE. (E) Relative levels of Flk-1, Gata1, Gata2, Lmo2, and SCL in day 3.25 EBs cultured in the presence or absence of Dox. Graph represents data from 2 independent experiments performed in duplicate. Bars indicate SE. *P < .05