Figure 1.
Dynamic translocation of the AChR into lipid rafts by agrin. A, Distribution of AChR in naive C2C12 myotubes. Triton X-100-treated lysates of C2C12 myotubes were subjected to sucrose gradient centrifugation. Twelve fractions were collected from top to bottom (1–12). Equal volumes of each of the 12 fractions were pooled together, which contain AChR in both raft and nonraft fractions and taken as 100% or input. Each fraction was subjected to Western blot analysis using indicated antibodies. GM1 was assayed by using HRP-labeled CTX. Representative blots from three independent experiments with similar results are shown. Right panels are standard curve blots showing various percentages of the input. Graphs at the bottom show protein and cholesterol content in the 12 fractions. Cav-3, Caveolin-3. B, Neural agrin-induced translocation of AChRs into lipid rafts. Myotubes were treated with 1 nm muscle agrin [Ag(0,0), 18 h], 1 nm neural agrin [Ag(4,8), 18 h], or 1 nm neural agrin for 12 h in the presence or absence of 2 nm neuregulin (NRG). Fractionation was done as in A. The amount of AChRs in lipid rafts (fraction 4 and 5) was calculated against the standard curve of different percentages of the input. *p < 0.01, Student’s t test. C, AChR clusters colocalize with GM1 in C2C12 myotubes. Fully differentiated myotubes were incubated with 1 nm neural agrin to induce AChR clusters, and myotubes were incubated with Alexa 594-conjugated α-BTX to stain the AChR and FITC-CTX to label GM1. D–F, Enrichment of the lipid raft marker GM1 at the NMJ. Adult rat diaphragm (D) and denervated leg muscle (E, F) sections were stained for GM1 using FITC-CTX and AChR using Alexa 594-conjugated α-BTX (D, E) or for synaptophysin (F). Scale bars: C, D, 20 μm; E, F, 50 μm.