LRP does not alter the secretion of Aβ in cultured cells. LRP^+/– and LRP^–/– fibroblasts transfected with human APP695 were generated as described. (a) To determine whether LRP levels contribute to changes in production and secretion of Aβ, APP-transfected LRP^+/– and LRP^–/– cells were metabolically labeled with ³⁵S-methionine for 20 minutes and immunoprecipitated with an Ab specific for APP (upper panel). In parallel experiments, medium was conditioned for 24 hours, and the amount of secreted Aβ was analyzed by immunoprecipitation (3134), followed by Western blotting (26D6) (lower panel). (b) To distinguish between Aβ secretion and LRP-dependent Aβ clearance within a 24-hour period, APP overexpressing LRP^+/– and LRP^–/– cells were treated with or without RAP (1 μM), and the medium was quantitated for the amount of Aβ by ELISA. Graph shows a representative experiment (n = 3) with means and SEM. (c) Confluent cultures of APP overexpressing LRP^+/+ and LRP^–/– fibroblasts were incubated with or without activated α2M (100 nM) in serum-free medium to induce uptake of Aβ through LRP. RAP (1 μM), an antagonist for all known LRP ligands, was added to block α2M-LRP–mediated effects. After 48 hours, medium was collected and analyzed for levels of Aβ by sandwich ELISA assay. Aβ levels are normalized to the no-treatment group of each cell line. Experiments were performed three times in triplicate. Error bars represent SEM.