|
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.