FIGURE 2.

Sublethal concentrations of exogenous Aβ(25–35) and Aβ(1–42) down-regulate DNA-PK activity. A and B, PC12 cells were treated with Aβ(25–35) (black columns) and Aβ(1–42) (white columns) at different doses (1–50 μm) for 24 h (A) and for different times (50 μm, 4–24 h, B). After treatments, 400 μg of WCE were subjected to dsDNA-cellulose pulldown, and a phosphorylation assay was performed. Values represent average ± S.E. of kinase activity with respect to untreated cells (100%). Asterisks identify p < 0.05 versus controls. C, 40 μg of the same protein samples were used for Western blot analysis of DNA-PK complex protein levels (DNA-PKcs, Ku70, and Ku86). Actin was used as loading control. D and E, densitometric quantitation of the immunoreactive bands corresponding to DNA-PKcs. Values represent the normalized percent changes in DNA-PKcs protein levels with respect to control (100%) after exposure to Aβ(25–35) at different doses (1–50 μm) for 24 h (D) and for different times (50 μm, 4–24 h, E) (average ± S.E.; n = 3). Asterisks identify p < 0.05 versus control values. F and G, PC12 cells were exposed (+) or not (−) to 50 μm aggregated Aβ(25–35) (F) or treated with 1 and 10 μm DOX (G) for 24 h and then subjected to biochemical fractionation to isolate nuclear (Nuc) and cytoplasmic (Cyt) proteins. Fractions were immunoblotted with antibodies for Ku70, Ku86, cytosolic marker α-tubulin, and nuclear marker Histone H1. Blots in the figure are representative of three independent experiments.