Figure 13

Aβ generation is altered in a cell cycle-dependent manner: H4-15X cells were synchronized by serum starvation and stimulated with serum containing media plus or minus olomoucine, roscovitine, aphidicolin, nocodazole, vinblastine, or taxol. Cell culture supernatants (A and C) and cell extracts (B and D) were immunoprecipitated and western blotted using 6E10 antibody. Control was performed similarly to the rest of the samples, except no primary antibody was used in immunoprecipitation assay. Cell culture supernatant showed a time dependent increase in Aβ generation upon serum stimulation (A and C). The extracts showed similar results which was visible only after longer exposure (D). Panels C and D represent longer exposure of the bottom part of the blots shown in A and B to show the Aβ levels in cell extracts. In both the cases roscovitine treatment was associated with a decrease in the level of secreted and cellular Aβ. Secreted APP was not altered in the supernatant although the level of APP and C-APP fragments are increased in the extracts prepared from nocodazole and vinblastine-arrested cells. Panel E and F show mean percent of Aβ (compared to secreted and full length APP) from 3 independent experiments under different treatment conditions. Data that showed significant changes are marked using a star (P < 0.05).