Figure 2

p23 depletion affects APP maturation and secretion in HeLa cells. A, Lysates of HeLa cells transfected with siRNA were probed with antibodies specific for p23, APP, calnexin, and Grasp55. Arrowheads indicate mature glycosylated APP. B, HeLaSwe cells were labeled with [³⁵S]met/cys for 3 h and APP FL and sAPP were immunoprecipitated from cell lysates and conditioned media, respectively. C, The levels of sAPP in 3 h-labeled conditioned media were quantified and normalized to APP synthesis determined by 15 min pulse labeling. AU, arbitrary units. D, HeLaSwe cells were pulse-labeled for 15 min and chased for the times indicated. APP FL was immunoprecipitated from cell lysates and signal intensities of immature and mature APP at each time point were quantified by phosphorimaging and normalized to APP synthesis. E, The levels of cell surface APP were quantified by mAb P2-1 binding of live cells as descried under Methods. The graphs in C and E represent mean ± SEM of three sets of transfections (p < 0.001), and D represents the mean of two experiments.