Figure 2

Sphingosine kinase-1 phosphorylation induced by insulin-like growth factor-1 is mediated by ERK 1/2. (A) Western blot analysis was performed using specific anti-phospho-sphingosine kinase (SK)1 antibody in membrane and cytosolic fractions prepared from serum-starved myoblasts treated with 50 ng/ml insulin-like growth factor-1 (IGF-1) for the indicated time intervals. A blot representative of at least three independent experiments with analogous results is shown. The histogram represents densitometric analysis of three independent experiments. Data reported are expressed as -fold increase of the membrane:cytosol ratio. The increase of phospho-SK1 membrane content induced by IGF-1 was statistically significant by Student’s t test (*P < 0.05). (B) Serum-starved myoblasts were pre-incubated for 30 minutes with ERK1/2 specific inhibitor (5 μM U0126) before being challenged with 50 ng/ml IGF-1 for 10 minutes. Cell lysates were separated by SDS-PAGE and immunoblotted using specific anti-phospho-SK1 and anti-β-actin antibodies. A blot representative of at least three independent experiments is shown. In the histogram band intensity corresponding to phosphorylated SK1 was normalized to β-actin and reported as mean ± SEM of three independent experiments, -fold change over control (set as 1). SK1 phosphorylation in IGF-1-challenged cells was significantly reduced by U0126 pre-incubation (Student’s t test, #P < 0.05).