Figure 4

p23 knockdown APP maturation and secretion in HeLa cells. A, Lysates of HeLa cell lines stably transfected with p23 or control shRNA plasmids were probed with antibodies specific for APP and p23. Quantitative analysis of sAPPα in the media conditioned by each stable cell line was performed using ELISA. The data represent mean ± SEM of three sets of samples; p < 0.001. B, Membranes prepared from two control and four p23 knockdown HeLa cell lines were incubated in vitro with C100 FLAG substrate and Aβ₄₀ generation was quantified by ELISA. C, The strategy for capturing active γ-secretase using the active-site γ-secretase inhibitor Merck C. D, The structure of Merck C. E, Solubilized HeLa S3 cell membranes were incubated with biotinylated γ-secretase inhibitor Merck C in the absence or presence of excess L-685,458. Active γ-secretase complexes were captured using streptavidin beads and analyzed by immunoblotting.