Inhibition of metap2 in zebrafish embryo affected N-methionine excision and erk phosphorylation. (A) GAPDH pI shifting assay showing Met-excised gapdh protein with a pI of approximately 8.4 and Met-unexcised GAPDH in metap2^MO or fumagillin-treated embryos with a lower pI at approximately 8.2. Met-excised gapdh protein with a pI at approximately 8.4 was recovered in metap2^MO rescued by metap2 mRNA. (B) Western blot comparing the level of total and phospho-erk1/2 between control, metap2^MO, and fumagillin-treated embryos normalized with β-actin. (C) Whole-mount immunostaining of phosphor-erk in Tg(fil1:gfp) at 48 hpf (control, 0 of 99 embryos showed ectopic erk phosphorylation; metap2^MO, 71.4% ± 3.2% in total 98 embryos showed ectopic erk phosphorylation; n = 4). Red indicates phospho-erk signal; green, endothelial cell; and yellow, phospho-erk signal colocalized with endothelial cell. Bars represent 50 μm