Figure 1

Myoblasts require glypican-1 expression for proper hepatocyte growth factor signaling. (A) Wild-type (WT) C2C12 myoblasts and C6 myoblasts (glypican-1-deficient clone) transiently transfected with rat glypican-1 (C6-Gly), were serum-starved for 6 hours and then treated with the indicated concentrations of hepatocyte growth factor (HGF) for 5 minutes. The cell extracts were analyzed by immunoblotting for total HGF receptor (Met) levels, phospho-Met (Tyr 1235/1349), phospho- and total AKT levels, phospho- and total levels of extracellular signal-regulated kinases 1 and 2 (ERK1/2), glypican-1 core protein (after heparitinase treatment), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tubulin. Total Met, AKT and ERK1/2 were used as loading control of its respective phosphorylated forms. GADPH and tubulin were used as loading control of glypican-1 expression levels. The Western blot images are representative of three independent experiments. (B) Quantitation of phospho-Met, phospho-AKT and phospho-ERK1/2 from three independent experiments is shown. Values are expressed as mean ± standard deviation. Statistical significance was assessed using two-way analysis of variance and a Bonferroni multiple-comparisons posttest. *P < 0.05, **P < 0.01. (C) Cell surface proteins of WT and C6 myoblasts labeled with EZ-Link Sulfo-NHS-Biotin (Pierce Biotechnology) as described in Methods. Aliquots of the cell extracts containing equal amounts of protein were precipitated with streptavidin-sepharose beads. The bound material was analyzed by Western blot immunoblotting against total Met. Aliquots of each assay obtained prior to the precipitation were analyzed by Western blot immunoassay for total Met, with tubulin used as the input control. Molecular weight standards are shown at left.