Fig. S3

A. Generation of rabbit antibodies against the zebrafish VWA domain. Lane 1, 10% SDS-PAGE analysis of the purified recombinant VWA domain produced in 293-HEK cells - The band migrates at the expected position. Lane 2, Western-blot analysis of 293-HEK conditioned media using the polyclonal antibodies obtained after rabbit immunization with the recombinant VWA domain (anti-COLXXII). B. Western-blot analysis of recombinant fulllength zebrafish collagen XXII incubated with (+) or without (-) collagenase as indicated. Zebrafish col22a1 cDNA was subcloned into the XhoI-XbaI sites of the pCS2+ vector (Turner and Weintraub, 1994) and the construct was used to transiently transfect 293-HEK cells. In absence of collagenase, COLXXII antibodies recognize the zebrafish full-length recombinant COLXXII protein as a band migrating at the expected molecular mass of the protein (200 kDa, lane 1). The collagenase digestion product revealed a 70 kDa fragment that corresponds to the undigested non-collagenous N-terminal domains, VWA plus TSPN (lane 2). A, B: protein standard size markers on the left. C-D. Immunofluorescence analysis of collagen XXII expression in adult zebrafish. Frozen cross section (C) and lateral section (D) stained with anti-COLXXII. Bars = 20 μm. E-F. Whole mount confocal immunofluorescence staining of 5dpf wild type larvae with anti-COLXXII (E) or anti-COLXII (F), actin is detected with phalloidin-rhodamine (red) and nuclei are stained with Hoechst-33344 (blue).