Figure 5

Glypican-1 is required to sustain the hepatocyte growth factor-dependent signaling in lipid rafts. Wild-type (WT) myoblasts were transiently transfected with an empty vector as the control or with a non-lipid-raft form of glypican-1 containing the extracellular domain of rat glypican-1 and the transmembrane and cytoplasmic domains of mouse syndecan-1 (F-GlySyn) [36]. C6 myoblasts were transiently transfected with an empty vector as the control or with rat glypican-1 (C6-Gly). Forty-eight hours after transfection, the cells were serum-starved for 6 hours and then treated with the indicated concentrations of hepatocyte growth factor (HGF) for 5 minutes. (A) The cell extracts were analyzed by immunoblotting for total HGF receptor (Met), phospho- and total Akt and phosphorylated extracellular signal-regulated kinases 1 and 2 (phospho-ERK1/2) and total ERK1/2. Glypican-1 core protein levels after heparitinase digestion of endogenous and both transfected forms of glypican-1 were detected by using an anti-glypican-1 antibody. Tubulin levels were used as loading controls. (B) Quantification from two independent experiments is shown. Statistical significance was assessed using two-way analysis of variance and a Bonferroni multiple-comparisons posttest. *P < 0.05, **P < 0.01.