Deregulation of SCL/Gata transcription factors in RapGEF2^−/− cells. (A) Differences in the expression level of hematopoietic marker, Brachyury, Fgf5, Flk1, Gata1, Gata2, Rex1, Scl, Flt1, and Vegf, and of Rap1a, Rap1b, and RapGEF1 in RapGEF2^−/− E10.5 yolk sac cells compared with RapGEF2^+/+ yolk sac cells, by Q-PCR. (B) Western blots showing the expression of the transcription factors SCL, Gata1, Gata2, and Lmo2, and the proto-oncogene c-myb in RapGEF2^+/+ (lane 1) and RapGEF2^−/− (lane 2) E10.5 yolk sac cells. Tubulin was used as a loading control. (C) Representative Western blots showing the level of Rap1, Rap1GTP, and B-raf, and the results of a B-raf/Rap1 pull-down assay showing the amount of B-raf-bound Rap1 (fourth panel from top), and the expression level of ERK1/2 and phosphorylated pERK1/2 in RapGEF2^+/+ (lane 1) and RapGEF2^−/− (lane 2) E10.5 yolk sac cells. Tubulin was used as a loading control. (D-E) The expression level of proteins (intensity of protein bands) was quantified by Imagequant software and presented as percent expression in RapGEF2^−/− cells compared with RapGEF2^+/+ cells, where the expression level was set as 100%. Then, the statistical analysis was shown as histograms. (F) Drawing displaying the possible role of RapGEF2 in the development of hematopoietic lineage. In this model, SCL/Gata transcription factors are required for the development of hematopoietic lineage, and RapGEF2 might regulate SCL through the Rap1/B-raf/ERK pathway.