Figure 3. Human brain samples from MCI and LAD show increased expression of Ras, activation of GSK-3αβ, and phosphorylation of APP and tau. Brain lysates prepared from the superior frontal gyrus of MCI, LAD, and control NAD subjects were analyzed by Western blot using the indicated antibodies. To confirm disease state, membranes were probed with 6E10 antibody to detect both FL-APP and Aβ (A), PHF-1 antibody to detect phosphorylated tau (B), and Tau 1 antibody to detect total tau (C). D, Finally, blots were probed with antibody to GAPDH, which served as a loading control. E, The bar graphs represent the quantitative analysis of protein levels normalized to GAPDH. Additional sets of membranes were probed with Grb2 (F), Ras (G), P-ERK (H), and after stripping, nonphospho ERK antibody to detect total ERK expression (I) and P-Thr231 tau (J). K, The membrane was then probed with GAPDH antibody, which served as loading control. L, The bar graph represents the qualitative analysis of the normalized proteins. Analyses were also done using antibodies to P-GSK3αβ (M), total GSK3αβ (N), and P-Thr668 APP (O). P, Membranes were reprobed with GAPDH antibody, which served as loading control. Q, The bar graph represents the quantitative analysis of protein levels normalized to the corresponding total protein GSK3 or to GAPDH. Statistical analysis was performed using one-way ANOVA. The data represent the mean ± SEM; NAD, n = 4; MCI, n = 5; and LAD, n = 5; *p < 0.05.