Figure 2.

SNX17 knockdown decreased cell surface LRP4 expression, MuSK phosphorylation, and AChR aggregation. (A) Cell lysates were immunoprecipitated with antibodies against SNX17, LRP4, or control IgG. The LRP4 and SNX17 proteins in immunoprecipitation and input were analyzed by western blotting. (B) Western blotting and qPCR detection of SNX17 protein (n = 3/group) and mRNA (n = 9/group) expression, respectively, in lentivirus-infected cells. (C) 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. (D) 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. (E) 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 KD-con vs. SNX17 KD, # represents -LRP4 antibody vs. +LRP4 antibody. All data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ^### P<0.001. 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.