Figure 10.

APPβ is generated from APP_FL that is concentrated in the ER. NT2N cultures of ∼1 × 10⁶ cells were infected with recombinant SFV containing either wild-type APP695 or APP695_ΔKK constructs. The dilysine motif concentrates APP_FL to the ER by an efficient retrieval mechanism. Duplicate cultures infected with wild-type APP695 were treated with 20 μg/ml BFA for comparison. Under these conditions, the cells were metabolically labeled with [³⁵S]methionine for 16 h. Radiolabeled cell lysates and media were then immunoprecipitated with antibody 53 (for APPβ, A) and Karen (for APP_FL and APPα/β, B). Radiolabeled immunoprecipitates were used to expose PhosphorImager plates (72 h) for visualization of bands. Unlike APPβ produced under BFA inhibition, APPβ derived from APP695_ΔKK was modified and secreted into the medium.