Statistical significance was assessed using two-way LLY-507 analysis of variance and a Bonferroni multiple-comparisons posttest. (C) C2C12 myoblasts treated with or without 10 mM PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/22721384 MCD for 1 hour as described in (A) were lysed and fractionated in sucrose density gradients as described in Figure 3. The distributions of total Met and caveolin 1 (Cav-1) were determined by immunoblot analysis. In (A) and (C), the molecular weight standards are shown at left.as regulators of HGF-dependent responses have not been studied in depth. In the present report, we show that, in myoblasts, glypican-1 located in lipid raft membrane domains was required for maximum HGF-dependent signaling and cell migration in vitro and in vivo. We also show that glypican-1 appears as an essential cell-surface, low-affinity binding site for HGF, likely acting as a presenter or facilitator of HGF to its high-affinity Met binding site, where it is cofractionated with the known HGF coreceptor CD44 [34]. Glypican-1, Met and HGF formed an active signaling ternary complex in lipid raft membrane domains. WhetherGuti rez et al. Skeletal Muscle 2014, 4:5 http://www.skeletalmusclejournal.com/content/4/1/Page 10 ofFigure 5 Glypican-1 is required to sustain the 1944-12-3 custom synthesis hepatocyte growth factor-dependent signaling in lipid rafts. Wild-type (WT) myoblasts were transiently transfected with an empty buy Tranylcypromine (hemisulfate) vector as the control or with a non-lipid-raft form of glypican-1 containing the extracellular domain of rat glypican-1 and the PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/20660090 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 CS-5122 chemical information 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.phospho-Met is relocated from non-lipid-raft to lipid raft domains in response to HGF or whether Met is directly activated in lipid rafts, where it is stabilized, are still not known. Chimeric non-lipid-raft glypican-1 (F-GlySyn) also coimmunoprecipitated with Met, but not with the active form of the receptor or with HGF, indicating that localization of glypican-1 in lipid raft domains was unnecessary for the interaction between Met and the extracellular part of glypican-1, but was required for binding of HGF and subsequent receptor activation.Phorylated extracellular signal-regulated kinases 1 and 2 (phospho-ERK1/2) and total ERK1/2, and tubulin was used as a loading control.