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Figure 2—figure supplement 1. Evidence for p-Smad3 presence and function in OFT ECs at 24 hpf, but no evidence for p-Smad1/5/8 presence.

(A–B’) Confocal images showing Tg(12xSBE:EGFP) expression (white) in 24 (A) and 75 (B) hpf hearts. Green, ECs; white dotted lines outline the OFT. (C–D’) Confocal images of 75 hpf wild-type larvae immunostained for p-Smad3 (white, C) or showing Tg(12xSBE:EGFP) expression (white, D); white dotted lines outline the OFT; green dotted lines outline the ECs. (E) Schematics of a 36 hpf heart showing ECs inside (OFT ECs) and outside (heart ECs) the OFT quantified in Figure 2C. (F) Intensity ratio of p-Smad3 and p-Smad1/5/8 immunostaining (normalized to DAPI) between ECs and surrounding cells in the OFT at 24 hpf. Every dot represents the ratio of ECs vs surrounding cells in each embryo. (G–H’) 24 hpf Tg(kdrl:EGFP) alk5+/+ (G) and alk5-/- (H) hearts immunostained for p-Smad1/5/8 (white). Dotted lines outline OFT ECs. (I) Quantification of p-Smad1/5/8 immunostaining (normalized to DAPI) in ECs and surrounding cells comparing 24 hpf alk5+/+ and alk5-/- OFTs. (F, I) Plot values represent means ± SD; p values from t-tests. Scale bars: (A, A’, C–D’) 10 μm; (B, B’, G–H’) 20 μm.

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