FIGURE 5.

Effect of transnasal delivery of erythropoietin (EPO)/insulin-like growth factor-I (IGF-I) on Akt/glycogen synthase kinase (GSK)-3β phosphorylation in mouse forebrain/olfactory bulb. Six-month-old wild-type (A) or gp120-transgenic (B and C) mice received the indicated dose of EPO (in U/ml) and/or IGF-I (in ng/ml) dissolved in vehicle (10mM sodium succinate buffer, pH 6.2) via transnasal delivery. Mice were sacrificed 30 minutes (A), 10 minutes (B), or 30 minutes, 60 minutes, and 24 hours (C) after the last application of peptide. The forebrain (C) or olfactory bulb (A and B) was then dissected to prepare lysates. Proteins from individual samples were subjected to immunoblot analysis with anti–phospho-Akt antibody (pAkt) (A) or anti–phospho-GSK-3β antibody (pGSK-3β) (B and C). Blots were then stripped and reprobed with anti-Akt antibody (Akt) (A), anti-GSK-3β (B and C), or antiactin antibody for total protein as a loading control. By densitometric analysis, combined treatment with EPO+IGF-I resulted in an increase in Akt phosphorylation compared to EPO or IGF-I alone. EPO (50U) plus IGF-I (2,000ng) also increased GSK-3β phosphorylation in gp120-transgenic mouse brain (B and C). For densitometric quantification, the ratio of pAkt to total Akt (A) or the ratio of pGSK-3β to total GSK-3β (B and C) for each lane was normalized to vehicle-treated samples. Data are expressed as mean + standard error of the mean of 3 to 6 independent experiments. *p < 0.05, **p < 0.01 versus vehicle (analysis of variance followed by Tukey-Kramer test for multiple comparisons).