Fig. 8: Proximity between signalosome and endocytosis components.

a Western blot of lysates from FlpIn T-Rex HEK293 cells +/− Wnt3a before or after 24 h of tetracycline (TET) induction of wt or YYYF > A mutant BioID baits (representative of three independent experiments), probed with antibodies (α) as indicated on the right; ABC, activated β-catenin. b Volcano plot showing YYYF-dependent hits identified by BioID (black, >2x compared to wt); Y axis, statistical significance of values obtained from three independent experiments was determined by Student’s t-test comparing each identified protein to the LRP6-BirA* bait (in cases where no peptide counts were identified in a sample, the value was replaced with 1 for calculation of ratios); selected hits are labeled (colored, related to Wnt signaling; black, unrelated to Wnt signaling). c Total unweighted spectral counts (>95% probability) of LRP6-BirA* bait (black) and selected hits from 3 biological replicates (expt1-3); gray underlay, hits reduced >2x in each experiment. d–f CoIP assays as in Fig. 1 (representative for three independent experiments), following co-expression of wt or mutant LRP6-GFP with FLAG-Axin and/or GSK3β-HA as indicated. g ΔB- or LRP6m10-sensitive hits identified in two independent TurboID experiments (expt 1 and 2) with (+) or without 2 h of Wnt3a stimulation; shown are total unweighted spectral counts (>95% probability) of LRP6-BirA* bait (top) and selected Wnt signaling-related hits (underneath); gray underlay, hits that are reduced >2x compared to wt in both experiments by ΔB (deleting YxxY, YxxΦ) or LRP6m10 (see also Supplementary Fig. 11). h Key phospho-acceptor sites in Axin LIRup (S317-S328), LIRα (S359) or PRTxR (T374) and cognate kinases shown in (g) with Kinase Library (KL) scores >80 (gray underlay); *, primed phospho-acceptor sites. (i) Subunits of clathrin adaptor complexes identified in two independent TurboID experiments shown in (g); gray underlay, hits reduced >2x in both experiments.