Figure 4.
Interaction between DAPK1 and APP. (A, B) CHO cells stably expressing APP WT were transfected with pRK5-Flag, pRK5-Flag-DAPK1 or pRK5-Flag-DAPK1K42A for 12 h. Cell lysates were immunoprecipitated with rabbit anti-Flag or rabbit anti-APP antibody. The precipitated samples were analyzed by western blotting with mouse anti-Flag or mouse anti-APP antibody. Total lysates, corresponding to 1% input, were also analyzed by western blotting with same antibodies. Anti-actin antibody was used as a loading control. The blots are representative of three independent experiments. (C, D) Endogenous DAPK1 (C) or APP (D) in SH-SY5Y cells was immunoprecipitated with mouse anti-DAPK1 or rabbit anti-APP antibody, respectively. Immunoprecipitated DAPK1 and APP were subjected to western blot analysis with mouse anti-DAPK1 or mouse anti-APP antibody. Anti-actin antibody was used as a loading control and the blots are representative of three independent experiments. (E) Schematic representation of full-length DAPK1 and its truncated mutants. (F) CHO cells stably expressing APP WT were transfected with pRK5-Flag-DAPK1 or its truncated mutants. Cell lysates were immunoprecipitated with mouse anti-Flag antibody. The precipitated samples were analyzed by western blotting with rabbit anti-APP antibody. Total lysates, corresponding to 1% input, were also analyzed by western blotting with same antibodies. Anti-actin antibody was used as a loading control. The blots are representative of three independent experiments. Asterisks denote heavy chain. (G, H) SH-SY5Y cells were transfected with pRK5-Flag, pRK5-Flag-DAPK1 or pRK5-Flag-DAPK1ΔDD for 32 h. The cell lysates were subjected to western blot analysis to detect phosphorylated APP at Thr668 and total APP protein. Anti-actin antibody was used as a loading control. The levels of human Aβ40 in cell culture supernatants were determined by a solid phase sandwich ELISA assay. Each data point represents the mean ± standard error of three independent experiments (*P < 0.05; ANOVA/Dunnett’s test).