Fig. 5
Posttranslational modifications of PS protein
(A) PS proteins are not sulfated. COS-7 cells transfected with the PS-1 or PS-2 cDNA were labeled with either [35S]-methionine ([35S]-meth.) or [35S]-sulfate and immunoprecipitated with antibody BOS4627. Arrowheads indicate the putative PS monomers and dimers. Oligomers of higher molecular weight were revealed after longer exposure. (B) Pulse-chase experiment. COS-7 cells transfected with the PS-1 (upper panel) or PS-2 (lower panel) cDNA were pulse-labeled with [35S]-methionine for 25 min and chased in the presence of excess amounts of unlabeled methionine for the time periods indicated. Cell lysates were immunoprecipitated with antibody BOS4627. Arrowheads indicate the putative PS monomers and dimers. Oligomers of higher molecular weight were revealed after longer exposure. (C) Treatment of cells with tunicamycin. COS-7 cells transfected with PS-1 or PS-2 cDNA were labeled for 3 hr with [35S]-methionine in absence or presence of 10 µg/ml tunicamycin. PS proteins were immunoprecipitated from cell lysates with antibody BOS4627 and endogenous secreted APPS from conditioned media of the same cells with antibody B5. No shifts in the molecular weights of PS-1 or PS-2 were obtained, while APPS isolated from the media of the same cells revealed the expected shift by approximately 2 kD. Two APPS polypeptides were observed, most likely representing endogenous βAPP695 (lower band) and βAPP751 (upper band). (D) Treatment with N-glycosidase F. [35S]-methionine-labeled PS-1 and PS-2 were precipitated from lysates of transfected cells with antibody BOS4627, and secreted APPS was precipitated from conditioned media of the same cells with antibody B5. Immunoprecipitates were incubated for 16 hr at 37°C in absence or presence of 2 units of N-glycosidase F. No shifts in the molecular weights of PS-1 or PS-2 were obtained, while APPS isolated from the media of the same cells revealed the expected shift by approximately 2 kD. Two APPS polypeptides were observed, most likely representing endogenous βAPP695 (lower band) and βAPP751 (upper band).