Figure 3.

SNX17 overexpression increased cell surface LRP4 expression, MuSK phosphorylation, and AChR aggregation. (A) Western blotting and qPCR detection of SNX17 protein (n = 3/group) and mRNA (n = 9/group) expression, respectively, in lentivirus-infected cells. (B) Myotube cells were treated with 10 μg/mL anti-LRP4 antibody for 30 min to induce internalization of LRP4 protein on the membrane, following which the anti-LRP4 antibody was removed and the cells were cultured for 30 min, and then the content of LRP4 protein in the membrane protein was analyzed by Western Blotting. n = 3/group. (C) Myotube cells were treated with 10 μg/mL anti-LRP4 antibody for 30 min, after which the anti-LRP4 antibody was removed, the cells were incubated with 10 ng/mL Agrin for 16 h, and the expression of MuSK and p-MuSK in total protein was analyzed by Western Blotting. n = 3/group. (D) AChR clusters (red) on lentivirus-infected C2C12 myotubes were labeled with R-BTX after application of separate or mixed Agrin and anti-LRP4 antibody, the nuclei were stained with DAPI (blue), and the number of AChR clusters on myotubes was counted and the length of AChR clusters was analyzed by ImageJ software. n = 3/group; Scale bar = 50 μm. * represents SNX17 OE-con vs. SNX17 OE. All data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ^## P<0.01. AChR, acetylcholine receptor; EAMG, experimental autoimmune myasthenia gravis; LRP4, low-density lipoprotein receptor-related protein 4; MuSK, muscle-specific receptor tyrosine kinase; R-BTX, rhodamine-labeled bungarotoxin; SEM, Standard Error of Mean; SNX17, sorting nexin 17. ns, no significance.