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Fig. 5
Maintenance of pERK required expression of two tyrosines in the cytoplasmic domain of VCAP1X2. Immunofluorescent staining revealed reduced pERK expression levels in VCAP1X2 mutant embryos (B) compared to WT (A) at 96 hpf. Reduced pERK expression levels in heart ventricles of VCAP1X2 mutants could be restored by injection with ΔN (F) or FL (G) VCAP1X2 mRNA but not with SP (C), ΔN Y2F (D) or Y2F (E) VCAP1X2 mRNA (n = 15 per condition, N = 3). Dashed lines illustrate the ventricular myocardium. Erythrocytes in the ventricular chamber show non-specific staining from secondary antibody. Nuclei were stained with DAPI. Scale bar, 30 μm. v, ventricle. (H) Levels of pERK or total ERK in embryonic hearts isolated from Tg(myl7:EGFP; myl7:H2AFZ mCherry) (myl7), VCAP1X2 mutant (mut) or VCAP1X2 mutant injected with Y2F or FL VCAP1X2 mRNA were measured by Western blot analyses with β-actin as loading control. Cropped representative blots for each protein are shown. Uncropped blots are shown in Supplementary Fig. S4. All blotting was performed using the same experimental conditions. (I) Quantification of pERK/ERK expression ratio among different treatments (N = 3). Error bars indicate standard error. Quantitative data were analyzed by ANOVA with Bonferroni multiple comparisons. Treatments that are not statistically different (α = 0.05) from each other are labeled with the same letter.