Xenbase Image ID: 174948

Fig. 3. The XIAPT180A mutant shows decreased binding and ubiquitylation of TLE3. (A) XIAPT180A ubiquitylates TLE3 to a similar extent to wild-type XIAP in vitro. In vitro-translated HA–TLE3 was incubated in an in vitro ubiquitylation reaction containing recombinant E1/E2, ubiquitin, and XIAP or XIAPT180A. Ubiquitylated TLE3 was visualized by immunoblotting with anti-HA antibody. (B) XIAPT180A exhibits reduced capacity to ubiquitylate TLE3 in cultured human cells compared to wild-type XIAP. HEK293STF cells were transfected as indicated, lysed under denaturing conditions, and His-Ub-modified proteins isolated by nickel affinity chromatography. XIAP and TLE3 were detected by immunoblotting with anti-MYC and anti-HA antibodies, respectively. The asterisk indicates endogenous XIAP. WCL, whole-cell lysates. (C) XIAPT180A exhibits decreased affinity for HA–TLE3 compared to wild-type XIAP. HEK293STF cells were transfected for 24 h as indicated followed by incubation in the absence or presence of recombinant Wnt3a for 24 h. Lysates were collected and immunoprecipitated (IP) with anti-MYC antibody. Co-immunoprecipitated HA–TLE3 was detected by anti-HA antibody. 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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