Figure 3.

Effects of MuSK IgG4 antibodies on AChR clustering and MuSK phosphorylation on C2C12 mouse myotubes. (A) Myotubes form few spontaneous AChR clusters (upper panel—DMEM) but, after physiological stimulation with agrin, their number increase markedly (middle panel—agrin). However, when the myotubes are exposed to MuSK antibodies, agrin-induced AChR clustering is severely impaired (lower panel—MuSK IgG4). Images are taken with an Olympus IX71 fluorescence microscope at 20× magnifications. Scale bar represents 50 μm. (B) Example of western blots to analyze MuSK phosphorylation. On the left, blot probed for phosphorylated tyrosine residues; on the right, the same blot is stripped and reprobed for MuSK expression. MuSK corresponds to a band at 97 kDa. MuSK phosphorylation is typically not detectable in the presence of medium only (first column—DMEM). After incubation with agrin for 45 min, MuSK phosphorylation increases markedly (second column—agrin) but this is prevented in the presence of MuSK IgG4s (third column—MuSK IgG4). Unpublished work, similar to Koneczny et al. (2013) and Huda et al. (2020).