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. 2016 May 3;7:11430. doi: 10.1038/ncomms11430

Figure 5. In addition to its known role in Axin proteolysis, Tnks promotes Wg pathway activation through a novel mechanism of Axin regulation.

Figure 5

(a-b) Generation of transgenic flies with wild-type Axin at levels higher than those resulting from loss of Tnks-dependent Axin proteolysis. (a) Immunoblot analysis of lysates from embryos expressing either Axin-V5 or AxinΔTBD-V5 integrated at the attP33 or the attP40 site. Lysates were prepared from embryos collected at 0–2 h of development, before the onset of Wg expression. Levels of V5 from attP33 AxinΔTBD-V5 are higher than attP33 Axin-V5 when expressed under the same conditions. Integration of the wild-type Axin-V5 transgene at a different genomic site, attP40, results in levels that are higher than attP33 AxinΔTBD-V5. Kinesin was used as a loading control. (b) Quantification of relative levels of indicated protein from experiments shown in a. Error bars represent s.e.m. of three independent experiments. P=0.0348 (one-way ANOVA with Kruskal–Wallis test). (c) The hatch rate of embryos expressing attP40 Axin-V5, attP33 Axin-V5 or attP33 AxinΔTBD-V5 with the mat-Gal4 driver. By comparison with embryos expressing attP33 Axin-V5 or attP40 Axin-V5, the hatch rate is reduced by expression of attp33 AxinΔTBD-V5. At least 200 embryos of each genotype were analysed, as indicated. (d–i) An increase in wild-type Axin, above the levels resulting from loss of Tnks-dependent Axin proteolysis, is compatible with the rapid stabilization of Axin following Wg exposure. (d–f) In stage 9 embryos expressing attP40 Axin-V5 driven by mat-Gal4, Axin-V5 accumulates in segmental stripes (brackets) that overlap the Wg stripes (asterisks). (g–i) Co-staining with V5 and Arm antibodies shows that Axin-V5 accumulates normally in Wg-responding cells, as revealed by co-localization with cells that accumulate Arm (white bars). (j,k) An increase in wild-type Axin, above the levels resulting from loss of Tnks-dependent Axin proteolysis, is compatible with the activation of Wg signalling. En expression is normal at both stage 8 (j) and stage 9 (k). A higher magnification view of the En stripes in k is shown (k*, white bar indicates the width of a single En stripe). Scale bar, 25 μm.