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Fig. S1 Knockdown of unc5b decreases levels of wild-type mRNA. RT-PCR analysis of control and morphant embryos shows that 1 ng unc5bSBMO1 or 4 ng unc5bSBMO2 significantly reduce levels of wild-type unc5b mRNA compared with uninjected embryos or to 8 ng control MO. Diagrams (top) show partial unc5b genomic structure (gray box, 5′ UTR; white boxes, coding exons; not to scale), with positions of MO sequences and RT-PCR primers indicated. Agarose gel images (below) show RT-PCR results for unc5b message and β-actin as a loading control. RT+/-, with or without reverse transcriptase. unc5bSBMO1 appears to abolish wild-type mRNA, while unc5bSBMO2 reduces wild-type mRNA and yields two additional bands. Sequencing of PCR products confirms that the upper band represents the wild-type product. The lower band corresponds to an in-frame deletion of amino acid residues 162-182, presumably reflecting a cryptic splice donor in exon 4. The middle band is a heteroduplex of the wild type and the alternate splice products. This deletion is in an Ig domain, which has been shown to be required for netrin binding (Kruger et al., 2004). DNA ladder sizes indicated in base pairs.