Figure 4

Extracellular signal-regulated kinase inhibition blocks extracellular nicotinamide phosphoribosyltransferase inhibition of insulin-like growth factor -1 signaling ( A), (B) Cells were pretreated with or without 10 μM mitogen-activated protein kinase kinase inhibitor (MEKi) for 30 minutes followed by treatment with extracellular nicotinamide phosphoribosyltransferase (eNAMPT) overnight, and insulin-like growth factor-1 (IGF-1) for 10 minutes or with IGF-1 alone for 10 minutes. After incubation, cell lysates were immunoblotted with phosphospecific antibodies to insulin receptor substrate-1 (IRS-1), AKT and extracellular signal-regulated kinase (ERK). Blots were stripped and reprobed with nonphosphospecific antibodies. Data are representative of at least three independent experiments. (C) The relative AKT (serine-473) phosphorylation level (normalized to total AKT protein) in the treated samples from three independent experiments was determined by densitometry analysis. Data presented as mean ± standard deviation.