Fig. 7. Lack of effect of filipin on the shedding of GPI-ACE. CHO cells transfected with either WT-ACE or GPI-ACE were incubated for 1 hour in the presence of PMA (1 µM) with or without filipin (10 nM). The conditioned medium was harvested and concentrated and the cells then fixed in paraformaldehyde and subjected to immunocytochemistry using the polyclonal anti-ACE antibody RP179 as described in Materials and Methods. (A) WT-ACE-transfected CHO cells incubated in the presence of PMA alone. (B) GPI-ACE-transfected CHO cells incubated in the presence of PMA alone. (C) WT-ACE-transfected CHO cells incubated in the presence of PMA and filipin. (D) GPI-ACE-transfected CHO cells incubated in the presence of PMA and filipin. (E) Conditioned media samples were assayed for ACE activity with BzGly-His-Leu as substrate. Results are the mean ± s.d. of three experiments.