Fig. 4

Cell cycle-dependent phosphorylation of Thr668 of APP

HeLa cells were synchronized at various cell cycle stages: G1 (synchronized by serum withdrawal and re-addition), S (synchronized with aphidicolin [Aph]), S (synchronized with hydroxyurea [Hu]), and G2/M (synchronized with nocodazole). APP was immunoprecipitated from cell extracts with G-369 antibody, samples were subjected to SDS-PAGE (6% acrylamide gel) and transferred to nitrocellulose membranes. (a) Upper panel: immunoblots were probed with pAbT668 or G-369 antibodies, [¹²⁵I]-protein A, and autoradiography performed. (b) Lower panel: cell cycle stages were determined by FACS scan analysis. Immunoblots shown in (a) were analyzed with a Molecular Dynamics PhosphorImager, and the relative levels of phosphorylated APP and total immature APP_770/751 calculated for each cell-cycle stage. Values for the G1 samples were set at unity. The data shown are the averages of two independent studies.