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Fig. 6. Processing of Dlg and Baz in extracts of embryos lacking DIAP1. Embryos lacking DIAP1 were collected either 1, 2 or 3 hours after cellularisation, and extracts were analysed on western blots using antibodies against Dlg (A,B), Baz (C) and DE-cadherin (D). Each blot was reprobed with an antibody against tubulin as a loading control (boxed bands). (A) Two isoforms are detected for Dlg in the WT. In th⁵ homozygous embryos, the amount of these isoforms markedly decreased and three shorter bands are detected (marked with asterisks), which are likely to be products of caspase cleavage of Dlg. Two of these products are still detectable in the th⁵ embryo extract 3 hours after the end of cellularisation. (B) Embryos overexpressing Reaper were collected 2 hours after the end of cellularisation on the basis of their phenotype and compared with wild-type-looking embryos from the same experiment, as well as th⁵ embryos. The same two Dlg isoforms are observed in wt, Reaper^OVER and th⁵ extracts, with an apparent MW of 120 and 100 kDa, respectively. Shorter bands of identical sizes are found in both th⁵ and Reaper embryos, indicating that they are both caspase digestion products. The apparent MWs of the putative caspase products are 85 kDa, 35 kDa (gels A and B) and 20 kDa (gel A). (C) A major band of apparent MW of 200 kDa is detected for Baz. The N-terminal antibody used for this experiment also recognises several smaller bands as previously reported (Wodarz et al., 1999). In th⁵ homozygous embryos, one main cleavage product is detected (apparent MW of 100 kDa) in addition to two minor ones (apparent MW of 80 kDa). (D) One major band of 120 kDa is detected for DE-cadherin in WT embryos. A minor band of 180 kDa is also found, which is thought to correspond to the precursor of DE-cadherin (Oda et al., 1994). The minor band is reduced in th⁵ embryos, but the levels of the major band remain the same. Moreover, we could not detect any cleavage products in th⁵ homozygous embryos. Together, these findings indicate that DE-cadherin is not processed by caspases in DIAP1 mutants.

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