Figure 3

Aβ42 levels are reduced in female but not male 5XFAD/BACE1 ^+/− mice. (A-C) Aβ42 dot blot assays were performed using GuHCl extracted brain homogenates from 4 (A), 6 (B) and 9 (C) month-old 5XFAD/BACE1^+/+ and 5XFAD/BACE1^+/− male and female mice (n = 6–17 mice per genotype per sex) and relative quantifications determined and presented as percentages of the mean female 5XFAD/BACE1^+/+ Aβ42 level. 5XFAD/BACE1^+/− females had ~30-40% less Aβ42 than 5XFAD/BACE1^+/+ females. In contrast, Aβ42 levels of male 5XFAD/BACE1^+/− and 5XFAD/BACE1^+/+ were indistinguishable, and both were significantly less than that of 5XFAD/BACE1^+/+ females. (D) To confirm the relative Aβ42 quantifications generated by the dot blot assay, Aβ42 levels in the brain homogenates from the 6 month-old cohort shown in (B) were quantified by commercial Aβ42 ELISA (Invitrogen), yielding very similar results. (E) Aβ42 measured by ELISA correlates significantly with Aβ42 measured by dot blot, further validating this method. (F) To determine the range and sensitivity of the Aβ42 dot blot assay, known quantities of synthetic Aβ42 were spiked into a transgene-negative BACE1^−/− homogenate, which was then GuHCl extracted and analyzed by dot blot (upper blot). Numbers above the blot indicate concentrations of Aβ42 in ng/mg total brain homogenate protein. (G) Aβ42 dot blot signals from (F) were corrected by subtracting the secondary antibody-alone background, converted to percentage of intensity of 1000 ng Aβ42/mg protein (y-axis), and plotted as a function of known Aβ42 concentration (x-axis). The dot blot is accurate through at least a 500-fold concentration range, with very good linearity from 1.9-250 ng Aβ42/mg total protein, as shown by the blue section of the curve (expanded in the inset). Error bars (SEM) are plotted in both graphs, but are too small to be seen except in the expanded inset.