Figure 3

Met is localized and activated by hepatocyte growth factor in lipid rafts by a glypican-1-dependent mechanism. C2C12 and C6 myoblasts were serum-starved for 6 hours and then treated without (control) or with 10 ng/ml hepatocyte growth factor (HGF) for 5 minutes. The cells were lysed with 1% Triton X-100 and fractionated by sucrose density gradients (5% to 45%). Twelve fractions were collected, but only the last ten fractions were analyzed (the lipid raft–enriched fraction started at fraction 4) by immunoblotting for total HGF receptor (Met), phospho-Met (Tyr 1235/1349), phosphorylated extracellular signal-regulated kinases 1 and 2 (phospho-ERK1/2), phospho-AKT and CD44. As fractionation controls, the presence of the lipid raft membrane protein marker caveolin 1 (Cav 1) and the non-lipid-raft domain marker Na⁺/K⁺-ATPase are shown. WT, Wild type.