Fig. 2

2-OST morphants display reduced levels of 2-OST protein and reduced levels of ³⁵S incorporation. (A) By western blot, levels of 2-OST protein were shown to be statistically significantly reduced by roughly half using both 2-OST MO1 and MO2 compared with WT and control embryos at 4 hpf. The antibody was validated using 2-OST RNA, with degradation product or altered post-translational modifications beneath the band, and 2-OST-EGFP RNA (two embryos/lane/sample, n=4 experiments). Quantification of protein levels was normalized to uninjected levels of 2-OST relative to the total levels of α-tubulin per sample. (B) WT, control MO and 2-OST MO embryos were injected with 1 nl 10 mCi ³⁵S, grown for five hours, and collected at 8 hpf (n=3, 90 embryos group/n). Protein purification was performed to isolate radiolabeled GAG chains and cpu per embryo was calculated based on liquid scintillation counting results. (C) Ratio of ³⁵S incorporation per embryo was normalized to WT. The 2-OST morphants exhibit a nearly 80% decreased rate of incorporation of ³⁵S compared with WT and control morphants. ^*P<0.05. Error bars represent s.d.