FIG. 6.

A single 5-min agrin pulse triggers long-lasting MuSK and AChR β phosphorylation. C2 myotubes were treated for 8 h continuously with 0.5 nM agrin. Parallel cultures were incubated for 5 or 40 min with 0.5 nM agrin, followed by agrin withdrawal, washing, and incubation in agrin-free medium for 8 h. All lysates were then split into two parts and were analyzed either by AChR (A) or by MuSK (B) precipitation, followed by phosphotyrosine immunoblotting (pTyr-blot). As controls, we added a 10 μM free α-BT (+T in panel A) or omitted the lysate or the MuSK antibody (−Ab in panel B). (A) Quantitation represents the means ± SEM of three experiments and shows no significant differences between the pulsed or continuous agrin treatments (P > 0.63). (B) Quantitation represents the means of two experiments that showed very small variations between each other. IP, immunoprecipitation.