Wnt effect on AChR clustering requires interaction with MuSK. A, Wnt9a and Wnt11, but not Wnt7a, bind MuSK in vitro. Flag-Wnts immobilized beads were incubated with MuSKect-Myc. Bound proteins were isolated, resolved by SDS-PAGE and blotted with antibodies against Myc and Flag. B, Wnt11 binds CRD of MuSK. Flag-Wnt11 immobilized beads were incubated with MuSKect, MuSKectΔCRD or MuSKCRD. Interactions were assayed as in A. *, p < 0.05, n = 3. C, Wnt-induced AChR clustering is rescued by MuSK, but not MuSKΔCRD, in MuSK-/- cells. MuSK-/- cells were transfected without or with respective constructs and resulting myotubes (identified by GFP encoded by the expression construct) were assayed for AChR cluster formation in response to agrin or Wnts as in Figure 1. Arrows, AChR clusters. *, p < 0.01, Student's t test. D, Wnt9a and Wnt11 induce MuSK tyrosine phosphorylation. C2C12 myotubes were treated by agrin or Wnts for 1 h. MuSK was purified by immunoprecipitation and probed with the 4G10 antibody. E, Wnt9a and Wnt11 induce MuSK dimerization. Flag-MuSK and MuSK-Myc were transfected into C2C12 myoblasts and resulting myotubes were treated by Wnts, agrin or control media for 1 h. Flag-MuSK was precipitated by Flag antibody and associated MuSK-Myc was determined by anti-Myc antibody. Band intensity of immunoblot was analyzed by ImageJ software. *, p < 0.01, one-way ANOVA with Student's t test.