Fig. S3

Functional relationship Clathrin with Dab2 in angiogenesis (related to Figure 3).
(A) CVP defects in cltca MO embryos. cltca MO injected embryos have multiple defects in vascular development, including severely disrupted CVP. Areas within rectangles are shown in higher magnification as insets. Scale bar is 200μm. (B) Efficacy of cltca MO was confirmed by RT-PCR. Total RNA was extracted at 48hpf to make cDNA and following primers were used for PCR reactions: Forward primer 5′-GGATCAACCCAGCCAACAT-3′, Reverse primer 5′-CTCGCACAGCGAAACAGAA-3′. (C) Blocking Cltca attenuated the extra vessel formation by Bmp2b over-expressing embryos. Scale bar is 100μm. The number of branches (N=3, total embryos=18, **p<0.001) and mean pixel intensity (N=3, total embryos=17, *p<0.01) within Bmp2b-induced ectopic vessels were drastically reduced in cltca MO injected embryos. Error bars represent standard error of the mean. (D) PLA was carried out to evaluate the co-localization of BMPRII with DAB2 and Clathrin. Scale bar is 10μm. (E) PLA to detect co-localization of DAB2 and BMPRII in the presence or absence of BMP6 (50ng/ml). Co-localization signal was not changed by presence of BMP6. Scale bar is 10μm.