Figure 8.

Mitosis-specific phosphorylation of APP: H4-15X cells were growth arrested by serum starvation for 48 hr and serum stimulated with and without roscovitine, olomoucine, or aphidicolin for 12 hr, and nocodazole, vinblastine, or taxol for 16 hr. Cell extracts were prepared and equal amounts of proteins were immunoprecipitated using 6E10 antibody and western blotted using P-APP antibody (A). B) APP levels in total lysate analyzed using 6E10 antibody. Panel C shows reprobe of blot B (without stripping) with actin antibody showing equal amount of proteins on gel. The histogram in panel D shows the percent of P-APP in cells under the different treatment conditions. The data represent the mean of 3 independent experiments with standard deviation shown. Cells arrested in metaphase showed significantly higher levels of P-APP (P < 0.05). Panel E shows the FACS analysis data from cells treated with cell cycle inhibitors. Cells were treated with roscovitine or aphidicolin for 12 hr or nocodazole or taxol for 16 hr and fixed and stained using propidium iodide before analysis on a FACS machine. Mean percent of cells in different phases of the cell cycle from 3 independent experiments is shown.