Figure 2.

Expression of wild-type hAPP695 in B103 cells inhibits p53 activation induced by UV or ST. (a) UV and ST induce nuclear translocation of p53 (confocal images of p53Ab1 immunostaining; ×100). (b) UV and ST increase p53 DNA-binding activity. Nuclear extracts were analyzed by EMSA with a ³²P-labeled p53 probe. The specificity of the p53/p53Ab1 DNA binding was confirmed in nuclear extracts from UV-treated untransfected B103 cells (Right) by competition assay using wild-type or mutant p53 oligonucleotides (36). (c) UV and ST increase luciferase activity in B103 cells transfected with a p53-responsive (p53⁺luc) or a p53-unresponsive (p53⁻luc) luciferase indicator construct. (d and e) Differential effects of wild-type and FAD-mutant hAPP695 on p53 DNA-binding activity and p53-responsive transactivation. (d) EMSA showing reduced levels of p53/p53Ab1 DNA-binding complexes in hAPP695wt-transfected B103 cells compared with mock- and hAPP695mut-transfected B103 cells. (e) Mock-, hAPP695wt-, and hAPP695mut-transfected B103 cells were cotransfected with p53⁺luc or p53⁻luc and treated with UV or ST. Luciferase activities in lysates of treated and untreated cells were assayed. Each column represents the mean of measurements obtained in six wells. Error bars indicate SD. ∗, P < 0.05; ∗∗, P < 0.01 by Tukey–Kramer posthoc test. Similar results were obtained in two additional experiments.