Fig. S3

Birc5a and Birc5b protein are both expressed maternally, but are functionally non-redundant. (A) Western blot of whole protein lysates from 20 mpa wild-type eggs (lane 1 and 2), and 40 mpf wild-type (lane 3) and motley embryos (lane 4). Anti-Survivin detects an ~25 KDa protein (lane 1) that is unaffected in motley mutants (data not shown), consistent with this antibody recognizing Birc5a (190 aa, predicted MW 22 kDa). Anti-Survivin-BIR detects a doublet of ~15 KDa protein in wild-type lysates (lanes 2, 3), which is affected in motley mutants (lane 4: lower band missing, upper band with reduced intensity). The mutant product encoded by the mutant motley allele is predicted to include the first 79 aa of the normal protein plus 32 novel aminoacids encoded by intronic sequence (111 aa total, MW ~13 kDa). Because aminoacid composition can affect protein mobility, determination of the precise identity of each band will require protein analysis. However, the data is consistent with anti-Survivin recognizing Birc5a and anti-Survivin-BIR recognizing Birc5b, and a lack of cross-reactivity between these two antibodies and their products. Total protein from each lysate loaded in each lane is indicated in μg. (B–J) Expression of Birc5a::GFP does not rescue the motley/birc5b cytokinesis phenotype. Stage IV oocytes from motley/birc5b mutant females were injected with birc5a::GFP mRNA and in vitro matured into eggs under the same conditions that allowed rescue through expression of birc5b::GFP mRNA. Injected oocytes expressed Birc5a::GFP at ~1hpi and matured into eggs (data not shown). Embryos derived from such Birc5a::GFP-expressing eggs activated normally upon contact with water following in vitro fertilization (B–D) but did not undergo cytokinesis (E–G). Labeling for α-tubulin and DAPI at a time equivalent to the 4-cell stage confirmed that such Birc5a::GFP-expressing, non-cleaving motley/birc5b embryos were indeed fertilized (H–J, note four nuclei showing normal karyokinesis).