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. 2012 Feb 15;32(7):2324–2334. doi: 10.1523/JNEUROSCI.4610-11.2012

Figure 7.

Figure 7.

Biglycan regulates agrin-induced MuSK phosphorylation. A, Biglycan potentiates agrin-induced MuSK phosphorylation. C2C12 myotubes were treated for 1 h with 1U agrin, 1.4 nm of purified recombinant biglycan, or both as indicated. The levels of MuSK tyrosine phosphorylation were then assessed by probing MuSK immunoprecipitates with anti-phosphotyrosine antibody (P-Tyr). The blots were stripped and reprobed with anti-MuSK to verify equal loading. Agrin induces MuSK phosphorylation and this activity is potentiated by biglycan. B, Quantification. Western blots were scanned and analyzed with ImageQuant software (GE Healthcare) to compare relative changes in MuSK phosphorylation. Simultaneous treatment with 1U agrin and 1.4 nm biglycan induces a 1.5-fold increase in MuSK phosphorylation compared with 1U agrin alone (p < 0.001, n = 10). Treatment with saturating concentrations of agrin (5 U) alone induces a 1.6-fold increase in MuSK phosphorylation above 1U agrin (p < 0.02, n = 5). C, Biglycan does not modulate MuSK phosphorylation induced by saturating concentrations (max) of agrin. C2C12 cells were stimulated with 5 U agrin in the presence or absence of 1.4 nm biglycan. MuSK phosphorylation was assessed as in A. D, Quantification. Biglycan does not induce a statistically significant change in MuSK phosphorylation above that induced by 5 U agrin alone (p > 0.3, n = 3). E, Equivalent levels of MuSK are expressed in C2C12 and bgn−/o myotubes. Cultured C2C12 and biglycan null myotubes were extracted and MuSK was incubated with anti-MuSK antisera followed by protein-A-beads. Immunoprecipitates solubilized in sample buffer, electrophoresed, transferred to nitrocellulose and probed with an anti-MuSK antibody. F, Biglycan is not required for agrin-induced MuSK phosphorylation. Biglycan null myotubes were treated for 15 min, 30 min, or 1 h with 1U agrin. MuSK activation was determined by probing MuSK immunoprecipitates with anti-tyrosine antibody (P-Tyr). Note that agrin induces MuSK phosphorylation in biglycan null cells within 15 min of treatment and phosphorylation levels increase through 1 h of treatment.