Fig. 4. Analysis of Sox9-AS specificity. (A) A Sox9 specific antibody recognizes only the major product of Sox9 in vitro translation reactions. (B) Western blot of Sox9 in vitro translated products. Sox9-AS blocked translation of Sox9 mRNA containing the 5'UTR target sequence (Sox9+5'UTR), but did not affect translation of Sox9 mRNA lacking the target sequence (Sox9). A nonspecific control oligo (Co-AS) had no effect on the translation of Sox9 mRNA derived from either construct. (C) Sox9 protein can be visualized by indirect immunofluorescence (green) in the nucleus (arrows) of the cells derived from animal explants injected with 1 ng of Sox9 mRNA at the two-cell stage. DAPI (blue) staining is shown for nucleus identification. (D) In vivo depletion of Sox9 protein. Detection of Sox9 protein in extracts from stage 17 embryos co-injected at the two-cell stage with 1 ng Sox9 mRNA (Sox9+5'UTR) and 10 ng of Co-AS or Sox9-AS. Tubulin is presented as a loading control.