Figure 8. APC2 lacking its Arm repeats cannot downregulate ßcat levels but retains some ability to blunt Wnt signaling.

A. The mutant proteins accumulate at near normal levels. Protein from embryo extracts expressing wild-type GFPAPC2, GFPAPC2ΔArmrepeats, or GFPAPC2Armrepeatsonly was immunoprecipitated with anti-GFP antibodies, separated by SDS-PAGE, and immunoblotted with anti-GFP antibodies. The expected transgenic proteins are indicated by red arrows and antibody heavy chain is also labeled. B–E. Representative cuticles from wild-type, and embryos maternally and zygotically null mutant for APC2, either alone or expressing the indicated transgene. Embryonic lethality and presence of adult escapers indicated below. B. Wild-type cuticle, showing alternating anterior cells secreting denticles (arrows) and posterior cells secreting naked cuticle (arrowheads). C. In APC2^g10 maternal/zygotic mutants, almost all cells are converted to posterior fates and only a few cells secrete denticles (arrow). D. APC2ΔArmrepeats restores alternately denticle belts (arrows) and naked cuticle (arrowheads), though denticle belts are often incomplete. E. In APC2^g10 maternal/zygotic mutants expressingAPC2Armrepeatsonly, most cells remain transformed to posterior fates and only a few cells secrete denticles (arrow). F. Quantification of rescue of Wnt signaling defects of embryos maternally and zygotically null mutant for APC2 by a GFP-tagged wild-type APC2 transgene (scoring scheme and wild-type rescue data from [36]), or by transgenes encodingAPC2ΔArmrepeats, or APC2Armrepeatsonly. G. Quantification of rescue of Wnt signaling defects of embryos maternally and zygotically double null mutant for APC2 and APC1 by either a GFP-tagged wild-type APC2 transgene (scoring scheme and wild-type rescue data from [36]) or by APC2ΔArmrepeats. H,I. Representative cuticles and embryonic lethality. Since the lethality of embryos expressing APC2ΔArmrepeats is higher than that of embryos with no transgene, this suggests additional embryos that are paternally-rescued may be dying, perhaps due to some dominant-negative activity of this protein on the paternally contributed APC2. Thus even the subtle degree of apparent rescue may simply reflect averaging in the less severe phenotype of these additional paternally rescued embryos. J. All transgenes are expressed and accumulate stably in SW480 cells. Immunoblot of cell extracts of human SW480 cells transfected with the indicated constructs. All of the APC2 constructs are N-terminally GFP tagged and detected with anti-GFP antibody. Tubulin serves as a loading control. K–M. SW480 cells transfected with the indicated constructs. GFP and ßcat. Arrows indicate transfected cells. K. SW480 cells, which are mutant for human APC, accumulate high levels of ßcat in their cytoplasm and nuclei (arrowhead). Transfection with fly APC2 rescues ßcat destruction (arrow). L. APC2Armrepeatsonly (arrow) does not rescue ßcat destruction or its nuclear localization. M. APC2ΔArmrepeats (arrow) does not rescue ßcat destruction but can retain some ßcat in the cytoplasm, lowering levels in nuclei (compare arrowheads). N. Only wild-type APC2 reduces ßcat levels, as quantified by Cellomics. O. Wild-type APC2 strongly reduces expression of the Wnt-regulated reporter gene, TOPFLASH, APC2ΔArmrepeats reduces TOPFLASH somewhat, and APC2Armrepeatsonly does not reduce TOPFLASH. Scale bars = 50 µm).