Fig. S1

Antisense αV MOs used in this study. (A) In the upper αV mRNA schematic, the relative locations of the translation-blocking antisense αV MOs are shown. In the lower αV mRNA schematic, the non-complementary AUG start site in modified, capped αV mRNA (mαV RNA) used for mRNA rescue is highlighted with a white box. (B) To assess the effectiveness of the αV1 MO, αV protein in zebrafish embryos was examined by western blotting of total zebrafish lysates. On the left, lysates from 5-8 somite stage (SS) embryos of uninjected (lane 1), control (2.5 ng; lane 2) and αV1 (1.25 ng; lane 3) were blotted with an affinity-purified rabbit polyclonal antibody raised against the extracellular domain of recombinant zebrafish αV (see Materials and methods). Quantification of αV protein levels in morphants relative to β-actin is shown on the right. Data represent mean ± s.e.m. of at least two independent experiments. (C) Exons 9 to 11 of the zebrafish αV genomic locus are represented with boxes and intronic segments with lines, not drawn to scale. The expected effect of the αVE110 splice-inhibiting MO is illustrated. (D) Schematic representation of putative wild-type αV and αV potentially transcribed in embryos injected with αVEI10 MO. Blue boxes 1 to 7 represent the putative amino-terminal 7-bladed β-propeller domain, and the gray box represents the putative transmembrane (TM) domain. Inhibition of splicing by αVEI10 MO would be predicted to result in a non-functional protein, which is truncated in the fifth blade of the β-propeller domain with an additional non-complementary 21 amino acid carboxy-terminal segment (green line). (E) Efficacy of αVEI10 MO on RNA splicing was assessed by RT-PCR analysis. Primers in αV amplified between exons 7 and 11 and yielded a 336 bp fragment in uninjected embryos (lane 1) or in embryos injected with 5 ng if an MO (β1bEI10 MO) designed to knock down the integrin β1b subunit (lane 2). By contrast, embryos injected with 5 ng of αVEI10 MO showed a predominant 283 bp fragment (lane 3) caused by deletion of exon 10. 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.