Fig. 1

Fig. 1 Western Blot Analysis of zebrafish Bruno-like (Brul) protein. (A) Schematic representation of the two isoforms of Brul proteins. Both forms possess three RNA recognition motifs (RRMs). The long isoform (GenBank Acc. No.BC049453) contains an additional 38 amino acid residues in its N-terminal region (gray box), whereas the short isoform has one extra amino acid residue in the RRM2 region (filled arrowhead), and four in the linker region (open arrowhead; GenBank Acc. No. AB032726), probably due to alternative splicing. (B) Two isoforms of Brul (approximately 55 and 60 kDa) are expressed in both male and female gonads. 50 μg of protein was loaded in each lane. The 60-kDa isoform is abundant in the ovary (lane 1), whereas no difference in the amounts of the two isoforms was observed in the testis (lane 2). No significant band was detected in the Western Blot with preimmune serum (lanes 3, 4). (C) Major two isoforms of Brul protein were observed throughout embryogenesis. Two phosphorylated proteins (approximately 57 kDa, filled arrowhead; and 62 kDa, open arrowhead) were also observed during early embryogenesis. Signal intensity at the shield and 12-hpf (6-somite) stages was relatively weak, consistent with faint signals in whole-mount immunostaining (see Fig. 2). Note that from the 32-cell to 12-hpf stages, the 55-kDa band is stronger than that at 60 kDa, whereas the 60-kDa band is stronger at the 24 hpf stage (lane 10). (D) Slower migrating bands disappear after CIAP treatment. Protein from blastomeres of 8-cell stage embryos was incubated at 37 °C in the presence or absence of CIAP. The 57-kDa protein, in particular, was almost completely eliminated. The asterisk represents CIAP protein.