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. 2020 Apr 9;5(7):e132023. doi: 10.1172/jci.insight.132023

Figure 5. Effects of agrin mutants on MuSK activation and MuSK phosphorylation.

Figure 5

(A) ATF2-luciferase reporter assay of HEK293 cells. HEK293 cells were transfected with ATF2-luc reporter and phRL-TK Renilla luciferase reporter plasmids with cDNAs for MuSK and LRP4. HEK293 cells were incubated with control conditioned medium (Control) or conditioned medium containing WT or mutant agrin-mycAP for 24 hours. Relative luciferase activity (RLA) was normalized for RLA without MuSK, LRP4, or agrin. Mean ± SD are indicated (n = 4 wells). Note that p.Y1877D reduced ATF-luc activity. One-way ANOVA followed by post hoc Tukey test. P < 0.05 is indicated by a single letter representing each group. (B) MuSK phosphorylation assay of C2C12 myotubes. C2C12 cells were added with control conditioned medium (Control), or conditioned medium containing WT or mutant agrin-mycAP for 2 hours. MuSK phosphorylation was detected by immunoprecipitation with an anti-MuSK antibody followed by immunoblotting with an anti-phosphotyrosine (pTyr) antibody. Quantification of phosphorylated MuSK is shown in the right panel. The ratio of phosphorylated to total MuSK was normalized to that of WT agrin. Mean ± SD (n = 3 independent experiments) are indicated. Note that p.R1509W and p.Y1877D reduced MuSK phosphorylation. One-way ANOVA followed by post hoc Tukey test. P < 0.05 is indicated by a, b, and c.