TGFβ and proinflammation signaling pathways are differentially regulated in spt5^m806 mutants. (A-B) Q-RT-PCR analyses of mutant and sibling controls at 36 hpf to compare proinflammation genes in whole embryos (A), and FACS-purified endothelial cells from Tg(fl1i:GFP) embryos (B). Gene expression is normalized to β-actin and presented as fold change relative to sibling controls (n = 3, mean ± SEM, *P < .05, **P < .01.). (C-D) Q-RT-PCR analyses of mutant and sibling controls at 36 hpf to compare TGFβ pathway gene expression in whole embryos (C), and FACS-purified endothelial cells from Tg(fli1:GFP) embryos (D). Gene expression is normalized to β-actin and presented as fold change relative to sibling controls (n = 3, mean ± SEM, *P < .05, **P < .01). (E) Western blot to compare Stat3-Jak2 (left) and TGFβ-Smad2/3 (right) activity between mutants and controls. Embryos (36 hpf) were used for protein extraction from the trunk (outlined by dashed lines in the embryo illustration) that contains the region where HSCs were generated. β-actin serves as the loading control. (F) Quantification of western results in panel E. Protein level is normalized to β-actin (n ≥ 3, mean ± SEM, *P < .05, **P < .01). TNF, tumor necrosis factor.