Extended Data Fig. 5: Schematic of experimental design to validate the function of mMymk.

a, Schematic of experimental design to validate in cis dimerization of mMymk by Proximity Ligation Assay (PLA). Created with BioRender.com. Two primary antibodies of different species were used to detect two signal peptide and flag-tagged (SF) mMymk monomers. The PLA probe binds the primary antibodies in close proximity to generate the signal. b and c, Trans proximity ligation assay (PLA) was performed on C2C12 myoblasts that express signal peptide Flag-mMymk and detected using mouse Flag (b) and rabbit Flag (c) antibodies. Representative images of PLA (red) with Flag (green) expressing mMymk^WT or mMymk^3M are shown. Nuclei were labeled with DAPI (blue). Scale bar, 10 μm. Experiments were repeated three times with similar results. d, Schematic of experimental design to validate antibody inhibition of mMymk by Ab^15G1. Created with BioRender.com. Ab^15G1 occupies mMymk dimers on either cell and would prevent mMymk interaction across cells, thereby inhibiting myoblast fusion. e, Schematic of experimental design to validate in trans interaction of mMymk by Proximity Ligation Assay (PLA). Created with BioRender.com. C2C12 myoblasts express either signal peptide and flag-tagged (SF) mMymk monomers or signal peptide and HA-tagged (SHA) mMymk monomers. Two primary antibodies (anti-HA antibody and anti-Flag antibody) from different species were used to detect two mMymk dimers in the neighboring cells. The PLA probe binds the close primary antibodies to generate localization of the signal. f, Representative images of HA (green) with DAPI (blue) expressing mMymk^WT or mMymk^3M on C2C12 myoblasts. The images show that HA antibody binds the two forms of mMymk^WT or mMymk^3M similarly. Scale bar, 10 μm. Experiments were repeated three times with similar results.