Fig. S5

Antisense β1b MOs used in this study. (A) β1b mRNA schematic; the relative location of the translation-blocking antisense β1b MOs is shown. (B) Exons 9 to 11 of the zebrafish β1b genomic locus are represented with boxes and intronic segments with lines; not drawn to scale. The expected effect of the β1bE110 splice-inhibiting MO is illustrated. (C) Efficacy of β1bEI10 MO on RNA splicing was assessed by RT-PCR analysis. Primers in β1b were amplified between exons 9 and 11 and yielded a 334 bp fragment in uninjected embryos (lane 1) or in embryos injected with 5 ng if an MO (αVEI10 MO) designed to knock down the integrin αV subunit (lane 3). By contrast, embryos injected with 5 ng of β1bEI10 MO showed 334 bp and 409 bp fragments (lane 2) caused by intron 10 insertion. Negative control reactions in the absence of any template (dH₂O, lane 7) or with total RNA extracts used as template showed no amplified PCR fragments. The following RNA templates were used as negative controls: uninjected embryos (lane 4); β1bEI10 MO-injected embryos (lane 5); and αVEI10 MO-injected embryos (lane 6). This experiment was repeated three times with similar results. (D) Schematic representation of putative wild-type β1b and β1b potentially transcribed in embryos injected with β1bEI10 MO. Brown boxes represent hybrid domains, which flank the βA domain (blue box); the putative four epidermal growth factor (EGF) repeats are shown with yellow boxes and the gray box represents the putative transmembrane (TM) domain. Inhibition of splicing by β1bEI10 MO would be predicted to result in a non-functional protein, which is truncated in the first EGF repeat.