Figure 2

Rspo2 is enriched at the NMJ and activates MuSK to induce AChR clustering.

(A) Rspo2 expression in the diaphragm and spinal cord normalized by Gapdh and also to E18.5 diaphragm. Mean and SD (n = 3) are indicated. (B) Rspo2 immunostaining and α-bungarotoxin staining for AChR at the NMJ of the tibialis anterior muscle (cross section). (C) AChR clusters visualized with α-bungarotoxin (red). C2C12 myotubes were cultured with 0.05 nM agrin and/or 0.05 nM Rspo2. Arrows point to the AChR clusters with an axis length of 4 μm or more. Blinded morphometric analysis is shown in Supplementary Fig. S2A. (D) ATF2-luciferase reporter assay to quantify agrin (10 ng/ml)- and Rspo2 (100 ng/ml)-mediated activation of MuSK signaling in transfected HEK293 cells. Relative luciferase activities (RLA) are normalized to that with empty vectors. Mean and SD are indicated (n = 3). **p < 0.01 by t-test. n.s., no significant difference. (E) Agrin- and Rspo2-mediated MuSK phosphorylation in C2C12 myotubes. Phosphorylated MuSK was immunoprecipitated (IP) and immunoblotted (IB) with indicated antibodies. (F) The ratio of phosphorylated (phosphoMuSK) and total MuSK was normalized to that with 0.1 nM agrin. Mean and SD (n = 3) are indicated. **p < 0.01 by t-test. The mean is also indicated in (E). BSA was added to control the amount of total proteins. (G) Additive effect of 0.05 nM agrin and 0.05 nM Rspo2 on MuSK phosphorylation in C2C12 myotubes. Phosphorylated MuSK was detected as in (E). Band intensities were normalized to that with 0.1 nM agrin (Supplementary Fig. S2B). The mean intensity is also is indicated below the blot. BSA was added as in (E). (H) Agrin- and Rspo2-mediated expression of rapsyn in C2C12 myotubes. Band intensities were normalized as in (G) (Supplementary Fig. S2C). The mean value is also indicated below the blot.