Adult expression of transgenic APP is independent of age at onset. A Cortical homogenates from juvenile- and adult-onset animals with 0 wk, 1wk, 2 mo or 6 mo of transgenic APP overexpression were immunoblotted with human-specific antibody 6E10 to determine whether timing of onset of APP induction differentially alters forebrain APP expression. The blot was overexposed to visualize the small amount of transgenic leak present in dox-treated mice (0 d, adult-onset). The outlined inset panel shows the same samples loaded with less protein per lane and a shorter exposure time. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was co-immunostained as a loading control. B A separate blot was immunostained with CT15; GAPDH was co-stained as a loading control. As in panel A, the intensity of GAPDH staining was lower in perinatal samples (0 d harvested at P0 and 1 wk harvested at P7) than in adult tissue although similar amounts of protein were loaded in each lane. The same GAPDH expression pattern was replicated with independent samples, and was observed with an unrelated control protein, SOD1 (not shown). C, D Quantitation of signal intensity for full-length APP from the 6E10 Western blot shown in (A) and the CT15 blot shown in (B). Because GADPH levels change over postnatal development, APP levels are plotted as absolute values rather than relative ratios. Graphs show mean ± SEM, n = 2 per time point for each age of onset. With just 2 samples per group, these values are intended only to highlight the difference in APP levels at the outset of expression (0 d). E Immunoblotting with 6E10 in a larger cohort of animals was used to obtain a more accurate measure of full-length APP expression after 2 mo and 6 mo of transgene expression. F Quantitation of APP intensity at 2 and 6 mo reveals that transgenic protein was lower in adult- than in juvenile-onset mice after 2 mo of expression (***p < 0.001), but reached similar levels at 6 mo (n.s.). Values are calculated relative to GAPDH, which expressed at stable levels in adult mice. Because separate blots were used to quantify APP levels at 2 mo and 6 mo, values for adult-onset were normalized to juvenile-onset at each age. Graph shows mean ± SEM, n = 6-9 per time point for each age of onset. White bars, juvenile onset; black bars, adult onset.