Ths-Pyr Chimeras highlight inhibitory activity of Ths C-terminus. (A) Schematics of ThsN-PyrC and PyrN-ThsC chimeric constructs and stage 11 embryos with 69B-GAL4 driving expression and function monitored with anti-Eve. Both chimeras give the +++Eve phenotype, meaning they support FGF activity. (B) Both chimeras are secreted, but cleaved differently in S2 cells. Supernatant immunoprecipitated with anti-HA and blotted with anti-HA, shows PyrN-ThsC is cleaved in a manner similar to Ths, while ThsN-PyrC may have both Pyr and Ths-derived cleavage sites. (C,D) Anti-Eve in stage 11 embryos with each chimera driven by ZenKr-GAL4 shows +++Eve cells in clusters outside the ZenKr domain for (C) pUASt-ThsN-PyrC and (D) pUASt-PyrN-ThsC. (E,F) Comparisons of Eve-positive cells per cluster in each hemisegment, scored and averaged as in Fig. 5. (E) ZenKr-GAL4 → ThsN-PyrC gives more Eve-positive cells than HA-Ths-Myc, suggesting that the Pyr C-terminus does not inhibit the Ths N-terminus the way that the Ths C-terminus does. (F) ZenKr-GAL4 → PyrN-ThsC has fewer Eve-cells than ZenKr-GAL4 → HA-Pyr1-348, and is similar to full-length HA-Pyr-Myc indicating the Ths C-terminus can likewise inhibit the Pyr N-terminus.
Tulin and Stathopoulos BMC Developmental Biology 2010 10:83 doi:10.1186/1471-213X-10-83