Figure 2.

NT2N neurons produce intracellular APPβ and Aβ. To demonstrate the presence of APPβ, samples of cell lysate and medium were collected from NT2N cultures and processed for immunoprecipitation (IP) with Karen, a polyclonal antibody that recognizes epitopes within the large ectodomain of APP. The presence of APPβ, APPα, APPα/β, and APP_FL was detected by immunoblotting (IB) with the corresponding antibodies (A). To show that Aβ but not p3 is produced intracellularly, NT2N neurons were radiolabeled with [³⁵S]methionine for 16 h. The cell lysate and the medium were then processed for immunoprecipitation with 4G8, a mAb that binds to both Aβ and p3, or Ban50, a mAb that recognizes only Aβ (B). Immunoprecipitates of Aβ and p3 were separated by electrophoresis in 10/16.5% step-gradient Tris-tricine gels. M, mature APP_FL; I, immature APP_FL.