FIG. 1.

Characterization of the mad2^EY mutant. (A) Schematic drawing showing the mad2^EY mutant caused by an EY element insertion into the third exon at bp 445 of the mad2 gene region (4). (B) The mad2^EY mutant can be maintained as laboratory homozygous stock with approximately 50%-reduced viability. This reduced-viability phenotype can be partially rescued by introducing an ectopically expressed transgene of gfp-mad2 (TM2-20) or mad2-Ypet (TM2-27; data not shown) on the second chromosome. The error bars indicate standard deviations. (C) Western blot results. (Lanes 1 and 2) GFP-Cdc20 protein in w⁶⁷ and mad2^EY-null mutant syncytial embryos, respectively. GFP-Cdc20 expression was around 2-fold that of endogenous Cdc20. (Lane 3) GFP-Mad2 and endogenous Mad2 expression in w⁶⁷ embryos. Lanes 2 and 4 show no detectable endogenous Mad2, or any of its truncated forms, from mad2^EY mutant embryo samples probed with an affinity-purified anti-full-length-DmMad2 antibody. (Lane 4) GFP-Mad2 was expressed at a level equivalent to that of the endogenous Mad2 in control samples (lane 3). Actin bands acted as loading controls. (D) (a) Schematic drawing showing the CBP-tagged fusion proteins used to test the anti-DmMad2 antibody. (b) Coomassie blue-stained 10% SDS-PAGE gel showing three purified fusion proteins, with molecule sizes indicated. The sample loading was as follows: lane 1, CBP-p27 (∼0.014 mg); lane 2, CBP-Mad2C (∼0.012 mg); lane 3, CBP-Mad2N (∼0.016 mg). (c) Western blot results showing that anti-DmMad2 antibody (Ab) can detect both the N- and C-terminal halves of truncated Mad2 fusions but not CBP-p27. Lane 4, CBP-p27; lane 5, CBP-Mad2C; lane 6, CBP-Mad2N. The samples loaded were 10 times less than those loaded for Coomassie blue staining. CBP-p27 (CBP plus full-length p27 protein) was used as the control to rule out nonspecific CBP signals.