Fig. 7

PEAK1 regulates VEGFR2 transcription through GATA2.

a WB of HUVECs treated with siRNAs, transfected or not transfected with a plasmid encoding GATA2, and stimulated with or without VEGF for 10 mins. Cells were cultured on collagen I coated plates. b Bar graph represents relative mRNA levels vs. HPRT1 by qPCR analysis of HUVECs treated as in (a). c Confocal images of ISVs at 32 hpf of Tg(fli1:egfp)^y1embryos with co-injection of gata2a mRNA or control mRNA and indicated MOs. Arrows point to stunted and disorganized endothelial sprouts. Scale bar = 50 μm. d Left bar graph represents the average ISV length of embryos treated as in (c). Mean ± SEM; n = 50. Right bar graph represents relative mRNA levels vs. zebrafish actb1 from the embryos treated as in (c) by qPCR analysis. Mean ± SEM; n = 3. HUVECs were cultured on collagen I-coated plates in (e) and (f). e WB of HUVECs transfected with siRNAs and treated with proteasome inhibitor MG-132. f, g Co-immunoprecipitated (IP) proteins from HUVECs with anti-GATA2 agarose were analyzed by WB for indicated proteins. In g, HUVECs were attached to plastic dishes with indicated coating. To compensate the protein level of GATA2 in Un group, the input was 1.8 folds compared to Fibro and Colla. h HUVECs were attached to plastic dishes with indicated coating and treated with different siRNAs. ECs were then treated with MG-132 for 6 h. Ubiquitination of IPed GATA2 protein was analyzed by WB. All data are representative of at least three independent experiments. ***P < 0.001; **P < 0.01; *P < 0.05; N.S. not significant