Fig. 4. NGF activates Rap1 and ERKs in a PKA and Src family kinase-dependent manner. (A) NGF activation of ERKs at late time points is Src-dependent in PC12 cells. PC12 cells were stimulated with NGF or EGF for the indicated periods in the presence or absence of PP2. Activation of endogenous ERKs (pERK1/2) was determined by western blotting. Total ERK2 levels are shown as loading controls. (B) Time course of Rap1 activation by NGF. PC12 cells were stimulated with NGF or EGF for the indicated times or left untreated (Un) and activation of endogenous Rap1 (Rap1-GTP) (upper panel) or ERK1/2 (pERK1/2, lower panel) was determined by western blot. Total Rap1 levels are shown as a loading control (middle panel). (C) PKA and Src are involved in Rap1 activation by NGF. PC12 cells were stimulated with NGF for 30 minutes in the presence or absence of H89 or PP2, or left untreated (Un). Activation of endogenous Rap1 (Rap1-GTP) was determined by western blot. Total Rap1 levels are shown as a loading control (lower panel). (D) PKA and Src-independent activation of Ras by NGF. PC12 cells were stimulated with NGF or EGF for 5 minutes in the presence or absence of H89 or PP2, or left untreated (Un). Lysates were analyzed for activation of endogenous Ras (Ras-GTP). Total Ras levels are shown as a loading control (lower panel).