Figure 1.
Treatment with mouse monoclonal-specific anti-ED-C99 antibody (mAbED-C99) markedly inhibits α-cleavage but promotes β-cleavage of human wild-type APP. (a) Human wild-type APP stably transfected CHO (CHO/APPwt) cells were plated in 24-well plates at 5 × 105/well and treated with mAbED-C99 at 0–1.25 μg/ml as indicated. (b) CHO/APPwt cells were treated with mAbED-C99, mAb22C11 (m22C11, recognizes APP66–81), or mAb2B3 (specifically and structurally recognizes sAPPα, but not full-length APP) antibodies, or isotype IgG1 control at 1.25 μg/ml. Immediately after 2 h treatment, cell supernatants were collected for western blotting (WB) analysis of sAPPα (using mAb2B3, upper panels) and Aβ secretion (using a monoclonal Aβ1–12 antibody BAM10, mAbBAM10, middle panels); cell lysates were prepared for WB analysis of APP processing products (using a polyclonal anti-C-terminal APP751/770 antibody, pAb751/770) and β-actin (internal control, lower panels). (c) Primary neuronal cells were cultured from cortical tissues of 1-day-old TgAPPwt mouse pups and seeded into 24-well-plates at 2 × 105/well for 18 h. These primary neuronal cells were treated with mAbED-C99 or IgG1 isotype control at 1.25 μg/ml for 2 h and then cell cultured media were collected for WB analysis of sAPPα (left upper panel) and secreted Aβ (left lower panel) levels as indicated; cell lysates were prepared for WB analysis of full-length APP (holo APP), APP-CTFs (right upper panel), and β-actin (right lower panel). These WB data are representative of four independent experiments with similar results. (d) In addition, the cell culture media were collected from the separated primary cortical neuronal cells following 8 h treatment with mAbED-C99 or IgG1 isotype control at 1.25 μg/ml for Aβ1-40/42 and sAPPα-ELISA. The results were presented as ng of Aβ40/42 or sAPPα per mg of total intracellular proteins (mean±S.D.; ***P<0.001). These ELISA data are representative of three independent experiments with similar results