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Fig. 4 The nitric oxide synthase (NOS)/nitric oxide (NO) pathway is involved in cbs and cth knockdown-induced defects of brain vascular development. (A and B) Effects of cbs and cth knockdown on the NOS/NO signalling pathway. Summary of nos1, nos2a and nos2b RNA expression (A) and total NO production (B) in 3-days post fertilisation (dpf) embryos. (C and D) Rescue effect of nos2a mRNA on the brain vascular developmental defects in cbs and cth morphants. Summary of increased total NO production (C) and midbrain vessel density (D) in 3-dpf embryos coinjected with cbs or cth MO and nos2a mRNA in comparison with those injected with cbs or cth MO. Six embryos were examined for each group. (E and F) No effect of cbs and cth knockdown on the protein expression of p-ERK1/2, ERK1/2 and VEGF. Representative blots (E) and summary (F) of Western blotting data. Error bars, SEM. *P<0.05, **p<0.01 (unpaired two-tailed Student’s t-test).