MuSK phosphorylation assay and AChR clustering assay with C2C12 myotubes.

C2C12 myoblasts were seeded on a plate coated with collagen I (BD Biosciences) and differentiated in DMEM supplemented with 2% horse serum for 5 days. The differentiated myotubes were treated with conditioned medium containing WT or mutant agrin-mycAP for 2 hours to induce MuSK phosphorylation and for 12 hours to quantify AChR clustering. For the AChR clustering assay, cells were stained with 10 mg/mL Alexa Fluor 594–conjugated α-bgt (1:100, Invitrogen) to label AChR and were fixed with 2% paraformaldehyde. Fluorescence images were observed under an Olympus XL71 fluorescence microscope. The signal areas and intensities of AChR clusters were analyzed with MetaMorph software (Molecular Devices). AChR clusters with the axis length less than 4 μm were excluded from the analysis.