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. 2006 Aug 24;25(17):4050–4060. doi: 10.1038/sj.emboj.7601288

Figure 5.

Figure 5

Postsynaptic proteins associate with CRLMs in myotubes. CRLMs were prepared from wild-type myotubes (C2C12 or clones SW10 and SW5), and fractions of the discontinuous sucrose gradients were collected. Fractions 9–12 represent the bottom gradient step (45% sucrose) containing the total cell extract. Fractions 5–8 represent the 35% sucrose layer and fractions 1–4 the top layer (5% sucrose). (A) Fractions were analyzed by immunoblotting (α-tubulin, caveolin-3, flotillin-2) or dot blotting (ganglioside GM1). Fractions 4–6 contain CRLMs and α-tubulin served as a negative control. (B) Fractions were analyzed for the content of cholesterol, showing high enrichment in CRLM fractions 4–6 (n=4). (C) Protein assays of gradient fractions reveal the bulk of protein in the bottom fractions, illustrating the specificity of the CRLM preparation (n=4). (D) Fractions were subjected to immunoblotting, showing that MuSK, AChRs (β-subunit), rapsyn, SFKs, α-dystrobrevin-2 (α-DB-2) and β-dystroglycan (β-DG) all partition efficiently into CRLMs. (E) Blots as shown in (D) were quantitated by densitometric scanning. For each protein, the intensities of bands in CRLM fractions 4–6 were related to the sum of all fractions to quantify the percentage in CRLMs (n=number of experiments).