LRP mediates the clearance of both Aβ40 and Aβ42 through a receptor-mediated uptake mechanism. (a) CHO cells overexpressing APP751 with V717F FAD mutation were grown to confluency, and IS-CHO was collected for 48 hours. Conditioned medium was then added to confluent nontransfected LRP^+/– and LRP^–/– fibroblasts for 48 hours in the presence of 20 nM α2M, and Aβ40 and Aβ42 levels were measured by sandwich ELISA. Experiments were performed three times in triplicate. Error bars represent SEM. (b) To determine optimal Aβ uptake, mixtures of 0.1 nM ¹²⁵I-Aβ with 0, 1, 2, and 3 nM α2M were incubated overnight at 37°C, and the incubation mix was added to confluent LRP^+/– (filled squares) and LRP^–/– (open circles) fibroblasts for 24 hours. The medium was then collected and subjected to scintillation counting for γ radiation. Percentage of Aβ uptake reflects the proportion of counts lost from the input amount. (c) To determine whether Aβ uptake is subject to self-competition, a mixture of 0.1 nM ¹²⁵I-Aβ and 2 nM α2M was incubated overnight at 37°C, and the incubation mix was added to confluent LRP^+/– fibroblasts in the presence of increasing amounts of excess unlabeled α2M/Aβ complex for 24 hours (filled squares). In parallel experiments, 1 μM RAP was coincubated with the ¹²⁵I-Aβ/α2M mix (open squares). The medium was then collected and subjected to scintillation counting for γ radiation. Percentage of Aβ uptake is normalized to maximal Aβ uptake in the absence of unlabeled Aβ/α2M complex. (d) To determine whether Aβ uptake is subject to saturation, increasing amounts of ¹²⁵I-Aβ/α2M complex (mixed as before) were added to confluent LRP^+/– cells for 24 hours, and the medium was collected and subjected to scintillation counting for γ-radiation. Aβ uptake is calculated in femtomoles and represented in a log scale. All experiments were performed three times in triplicate, and a representative experiment is shown. Error bars represent SEM.