FIG. 6.

Glypican-1 is the only HSPG associated with lipid rafts. (A) C2C12 myoblasts were lysed and then fractionated in sucrose density gradients (5 to 45%). The 12 fractions collected were analyzed by immunoblotting for HSPGs, as explained in the legend of Fig. 1A, as well as the lipid raft membrane protein markers, GM-1 (ganglioside GM-1),. and Cav-1. Na⁺/K⁺ATPase (ATPase), were used as a nonlipid raft domain marker. On the left, the molecular weight standards are indicated in thousands. (B) Indirect immunocytolocalization analysis for glypican-1 (red) in C2C12 myoblasts treated with or without MβCD or PI-PLC. The nuclei were subjected to Hoechst staining (blue). The arrows indicate the punctuated pattern of glypican-1 on the cell surface, and the arrowheads point at the ECM-associated glypican-1. (C) C2C12 myoblasts were treated with MβCD or left untreated and then fractionated as described for panel A. The fractions were analyzed for glypican-1 and Cav-1 distribution by Western blotting. (D) As shown on the left, C6 clone myoblasts were transiently transfected with rat glypican-1 containing a FLAG epitope in its amino terminal (F-Gly) or the empty vector. After 48 h, the cells were fractionated as described for panel A. The 12 fractions were pooled into three groups: group I (fractions 1 to 4), group II (fractions 5 to 8), and group III (fractions 9 to 12). The fractions in each group were analyzed for the distribution of rat glypican-1 by using an anti-FLAG antibody. As shown on the right, in a parallel experiment, C6 myoblasts transfected with F-Gly were treated with MβCD or left untreated and then fixed and analyzed by immunofluorescence for the presence of the FLAG epitope. syn-3, syn-1, syn-2, and syn-4 represent syndecan-3, -1, -2, and -4, respectively.