Xenbase Image ID: 174947

Fig. 2. The XIAPT180A mutant exhibits decreased Wnt activity compared to wild-type XIAP in cultured human cells and in Xenopus embryos. (A) GSK3 associates with XIAP and the kinase mutant, XIAPT180A, to a similar extent. HEK293STF cells were transfected with vector, HA–GSK3, MYC–XIAP and MYC-XIAPT180A as indicated, whole cell lysates (WCL) were collected, and immunoprecipitation (IP) performed with anti-MYC antibody. The asterisk indicates endogenous XIAP. (B) The XIAPT180A mutant does not exhibit a localization that is distinct from XIAP. Left, HEK293STF cells were transfected with MYC–XIAP or MYC–XIAPT180A, fixed, and immunostained for MYC and DNA (DAPI). Scale bars: 12.5 µm. Right, cytoplasmic and nuclear fractionations were isolated from transfected cells and immunoblotted for MYC, Histone H3 (a nuclear marker) and GAPDH (a cytoplasmic marker). (C) In contrast to wild-type XIAP, XIAPT180A does not potentiate Wnt3a signaling. HEK293STF cells were transfected with vector, MYC–XIAP or MYC–XIAPT180A as indicated, for 24 h, and cells incubated in the absence or presence of recombinant Wnt3a for 24 h. The asterisk indicates endogenous XIAP. Graph shows mean±s.d. of TOPflash activation normalized to the cell titer. ***P<0.0001 (one-tailed Student's t-test). All experiments were repeated at least three times. (D) The XIAPT180A mutant does not potentiate Wnt8 (Xwnt8)-induced axis formation in Xenopus embryos, in contrast to wild-type XIAP. Embryos (four-cell stage) were injected ventrally with control, XIAPT180A or XIAP mRNA (2 ng each) with or without Wnt8 mRNA (0.1 ng) and allowed to develop. Representative Wnt8-, XIAP- and XIAPT180A-injected embryos are on the left. The percentage of embryos with secondary axis formation is shown on the right (n≥31 per group). ***P<0.0001 (Fisher's exact test, with Bonferroni correction). All results were replicated at least three times. Image published in: Ng VH et al. (2018) Copyright © 2018. Image reproduced with permission of the Publisher, The Company of Biologists Ltd. Larger Image Printer Friendly View

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