<p>(A) Domain organization of Scube3 encoded by the <i>Scube3-001</i> splice variant. This protein consists of a signal peptide sequence (S), nine EGF-like repeat domains, a spacer region containing three cysteine-rich repeats, a CUB domain and C-terminal sequence; (B) The murine <i>Scube3</i>-<i>001</i> splice variant incorporates 6699 base pairs and contains 22 exons. Exon 1 encodes the signal peptide sequence, exons 2–10 encode the nine EGF-like domains, exons 11–18 encode the spacer region and cysteine residues in proximity to the CUB domain, exons 19 and 20 encode the CUB domain itself and exons 21–22 encode the amino acid sequence present at the C-terminal region of the protein, downstream of the CUB domain; (C) The <i>Scube3-004</i> splice variant incorporates 4103 base pairs and contains 18 exons. The splice variants are aligned to each other, meaning that exon 1 of <i>Scube-004</i> corresponds to exon 3 of <i>Scube-001</i> and so forth. This is interrupted by exon skipping at exon 16 in <i>Scube-001</i>, such that intron 13–14 of <i>Scube-004</i> corresponds to intron 15–16, exon 16 and intron 16–17 of <i>Scube3-001</i> (a total region of 714 base pairs). Exon 18 of <i>Scube3-004</i>, which corresponds in part, to exon 21 of <i>Scube-001</i> is also modified by an alternative 5′ splice site, incorporating 1684 ‘extra’ base pairs from the adjacent intronic sequence (intron 21–22 of <i>Scube-001</i>). Red boxes represent exons, white boxes represent untranslated regions of exon 1 and 22; (D) The targeting vector contains a ZEN-Ub1 cassette consisting of a <i>lacZ-p(A)</i> reporter and <i>hUbCpro-neo-p(A)</i> selectable marker flanked by <i>loxP</i> sites, which replaces the whole <i>Scube3</i> coding region following homologous recombination in ES cells (A–D are not to scale); (E) PCR analysis of DNA extracted from tail samples using wild type and mutant pairs of primers for wild type and mutant alleles, respectively. The wild type set of primers recognize a 25 base pair intronic sequence situated between exon 1 and 2 (blue arrows) and amplify a DNA fragment of 190 base pairs; whilst the mutant pair of primers amplify a 605 base pair DNA fragment within the Neo cassette (green arrows). For each sample, two separate PCR reactions were run (1) 100 base pair DNA ladder; (2) Negative control; (F) Western blot analysis reveals an absence of Scube3 in protein lysate derived from <i>Scube3<sup>−/−</sup></i> embryos. Protein lysates derived from human osteoblasts and <i>Scube3<sup>WT</sup></i> mouse embryos were used as positive controls. α-tubulin was used as a loading control. As predicted, the Scube3 protein detected in human osteoblasts and wild type embryos was approximately 110 kDa in size.</p
