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

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(a) Use of a GST-WWE pull down assay for specific detection of ADP-ribosylated Axin. Detection of ADP-ribosylated mouse Flag-Axin1 and Flag-Axin1ΔTBD using either GST-WWE or GST-WWE^R163A control pull downs in HEK293T cells. Flag-Axin1 is pulled down by GST-WWE; however, the Flag-Axin1ΔTBD control is not. (b,c) The levels of ADP-ribosylated Axin rapidly increased following Wnt exposure in human cells. (b) HEK293T cells were treated with Wnt3A for 0.5 h, and cell lysates were subsequently pulled down by GST-WWE followed by immunoblot analysis with the indicated antibodies. Following treatment with Wnt, phospho-LRP6 is pulled down by GST-WWE. (c) Quantification of relative ADP-ribosylated Axin1 levels from experiments shown in (b). Error bars represent s.e.m. of three independent experiments. P=0.0062 (Student's t-test). (d,e) The level of ADP-ribosylated Axin rapidly increased following Wg exposure in Drosophila cells. (d) Immunoblot of lysates from S2R+ cells treated with Wg conditioned medium (CM) for 1 h and then subjected to GST-WWE pull down. Treatment with Wg CM increased the level of ADP-ribosylated Axin pulled down with GST-WWE. Following treatment with Wg CM, phospho-Arrow is pulled down by GST-WWE. (d, input) Treatment with Wg CM induced a mobility shift in Axin. (e) Quantification of relative levels of ADP-ribosylated Axin from experiments shown in (d). Error bars represent s.e.m. of four independent experiments. P=0.0147 (Student's t-test). (f–h) The levels of Axin-V5 and ADP-ribosylated Axin-V5 are rapidly increased after the onset of Wg stimulation in Drosophila embryos. Embryos at stages 4–5 and stage 9 expressing Axin-V5 driven by the mat-Gal4 driver. Stages 4-5 (1.5 to 2.5 h of development) are before the onset of Wg stimulation and stage 9 (3.5 to 4.5 h of development) is after the onset of Wg stimulation. Lysates were immunoblotted by indicated antibodies, followed by GST-WWE pull down. Axin-V5 and phospho-Arrow are pulled down by GST-WWE following Wg stimulation. (g and h) Quantification of relative Axin-V5 and ADP-ribosylated Axin-V5 levels from experiments shown in (f). (g) P=0.0321 (Student's t-test). (h) P=0.0305 (Student's t-test). Error bars represent s.e.m. of three independent experiments.