Fig 1: Anti-MuSKCRD antibodies specifically inhibit Agrin-induced AChR clustering in vitro.(A and D) WT primary myotubes were treated with Agrin (Ag) together with increasing concentrations of MuSKCRD-immunized rabbit serum (CRD, %) or were left untreated (UT) for 16 hours (A). MuSKΔCRD primary myotubes were subjected to similar treatment with the optimal immunized rabbit serum concentration (1%, D). Cells were stained with α-bungarotoxin to label AChR clusters. Scale bar: 10 μm. (B and E) Quantitative analysis of the number of AChR clusters per mm² of myotube for A and D. (C) Lysates from WT and MuSKΔCRD primary skeletal muscle cultures were subjected to IP with control IgG or anti-MuSKCRD antibodies. Whole-lysate Western blots (Input) were performed to demonstrate the presence of the proteins of interest. The specific MuSKΔCRD (85 kDa) band overlaps with a nonspecific signal observed in all lanes. IB, immunoblot; GAPDH, loading control. The data are shown as mean ± SEM. n = 3 independent experiments; at least 50 myotubes were analyzed for each treatment in each experiment. White-filled large symbols represent the mean of all the myotubes analyzed for each biological replicate; ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; 1-way ANOVA with Tukey’s post hoc test (B), 2-way ANOVA with Tukey’s post hoc test (E).
Fig 2: Molecular mechanisms of anti-MuSKCRD antibodies in vitro.(A) Western blot of surface-biotinylated proteins (Biot) in C2C12 myotubes with (CRD) and without (Ctl) treatment with anti-MuSKCRD antibodies for 16 hours. Whole-lysate Western blots (Input) were performed to demonstrate the presence of the proteins of interest. Ctl, control; IB, immunoblot; TfR, transferrin receptor; GAPDH, loading control. (B) Quantitative analysis of A showing the ratio of biotinylated MuSK to TfR normalized relative to Ctl conditions. (C) MuSK-FLAG and Lrp4-HA co-IP assay with FLAG Fab-Trap agarose in transfected COS7 cells. Cells were left untreated (Ctl) or were subjected to pretreatment with anti-MuSKCRD antibodies (CRD) for 16 hours. (D) Quantitative analysis of C, corresponding to the ratio of IP Lrp4-HA to MuSK-FLAG normalized against the Ctl condition (lane 4 in C). (E) MuSK IP in C2C12 myotubes left untreated or treated with Agrin after 3 hours of pretreatment with anti-MuSKCRD antibodies (CRD). The 4G10 anti-phosphotyrosine antibody was used to assess MuSK phosphorylation. (F) Quantitative analysis of E corresponding to the 4G10 signal divided by the IP MuSK signal normalized against Ctl conditions (Ctl untreated [UT]). The data are shown as mean ± SEM. n = 4 (B and D) and n = 3 (F) independent experiments; ns, not significant; *P < 0.05; **P < 0.01; ****P < 0.0001; Mann-Whitney U test (B and D) or 2-way ANOVA with Tukey’s post hoc test (F).
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