Effect of extracellular nicotinamide phosphoribosyltransferase on insulin-like growth factor-1 signaling. (A) Chondrocytes were treated with various concentrations (0 to 5.0 μg/ml) of extracellular nicotinamide phosphoribosyltransferase (eNAMPT) overnight and then stimulated with 50 ng/ml insulin-like growth factor-1 (IGF-1) for 10 minutes. Cell lysates were immunoblotted with phosphospecific antibodies to IGF-1 receptor (IGF-1R), insulin receptor substrate-1(IRS-1), AKT, and extracellular signal-regulated kinase (ERK). (B) Cells were stimulated with 5 μg/ml eNAMPT or 50 ng/ml IGF-1 for 0 to 60 minutes. After incubation, cell lysates were immunoblotted with total or phosphospecific antibodies to ERK. The immunoblots presented are representative of at least three independent experiments.