Fig 2: Determination of the domains responsible for the interaction between C9orf140 and Axin1. a Identification of the C9orf140 domain responsible for interacting with Axin1. The 293T cells were transfected with Myc-Axin1 and a series of Flag-tagged C9orf140 deletion mutants as depicted in the diagrams. Cell lysates were immunoprecipitated with an anti-Flag antibody, and the precipitates and whole-cell lysates were then analyzed by western blotting. b Identification of the Axin1 domain responsible for interacting with C9orf140. Flag-C9orf140 was co-transfected into 293T cells with Myc-Axin1 or a series of Myc-tagged Axin1 mutants. Flag-C9orf140 was immunoprecipitated, and the precipitates and cell lysates were then subjected to western blot analysis

Fig 3: ADP-ribosylated Axin rapidly accumulates following Wnt stimulation.(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-Axin1?TBD using either GST-WWE or GST-WWER163A control pull downs in HEK293T cells. Flag-Axin1 is pulled down by GST-WWE; however, the Flag-Axin1?TBD 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.

Fig 4: Quantitative proteomic analysis of TNKS DKO cells. a Immunoblot of HEK293T TNKS KO cell lines demonstrating stabilization of Axin1 and reduction of ß-catenin in TNKS KO cell lines. Protein levels of Axin and ß-catenin in total cell lysates from KO cell lines relative to tubulin and normalized to WT cells are indicated below the blot. b Experimental strategy for analyzing protein abundance in TNKS DKO vs. WT cells using TMT labeling. c Results of protein abundance analysis. d Volcano plot indicating the 608 proteins showing a significant change in abundance (see Supplementary Data 1). e Heat map indicating 23 of the 74 proteins that are stabilized in TNKS DKO cells with a tankyrase binding site indicated on the right; additional sites (if present) are noted in Supplementary Data 2. Note, Axin1 has a non-canonical site, RxxVxGxE. Gene names in bold were selected for further analysis

Fig 5: Rapid appearance of Axin in segmental stripes following Wg exposure.(a–l) Axin is distributed uniformly in the embryonic ectoderm before and at the onset of Wingless (Wg) expression. Confocal images of embryos expressing Axin-V5 driven by the mat-Gal4 driver. Genotype left, antibodies top. Embryonic stage and developmental time in hours after egg lay (AEL) are indicated at the top right of a,g and m. Anterior left, dorsal up. In stage 5 and 6 embryos, Axin-V5 and the transmembrane protein Neurotactin are distributed uniformly throughout the ectoderm (a–l). Higher magnification images (d–f and j–l). (m–r) Axin is distributed in segmental stripes after the onset of Wg expression. By stage 9, Axin-V5 accumulates in segmental stripes with increased staining intensity (m–o). Higher magnification images (p–r). White bars indicate the width of a single Axin stripe. Scale bar, 25 µm.