Fig. 4
Levels of cell-surface β PP after PDBu
(A) Immunoprecipitation of full-length βPP with CT15 antibody immediately after surface radioiodination. The amount of radiolabeled βPP was reduced 15 min after PDBu pretreatment. Molecular weights determined from prestained markers are shown on the right. (B) Levels of cell-surface βPP were measured by radioiodinated 1G7 monoclonal antibody binding. βPP-transfected CHO cells were pretreated with PDBu for 5, 10, or 30 min before incubating with radiolabeled 1G7 antibody. The results are expressed as percent radioactivity of untreated control cells measured at time 0 (average ± SEM). (C) Levels of newly synthesized βPP arriving to the cell surface were determined by selectively immunoprecipitating cell-surface βPP with 5A3/1G7 monoclonal antibodies added to intact cells. Transfected wild-type CHO cells were pulse labeled with 35S-methionine for 10 min, followed by chase periods of 10 or 20 min with or without PDBu. Treatment with PDBu (+) showed marked reductions in the amount of newly synthesized cell surface βPP (bracket) as compared with untreated control cells (−). The background is higher in this assay because the labeled lysates cannot be thoroughly precleared.