Supporting information for Ueda et al. (2003) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0631617100
Fig. 8.
Comparison of enzyme activity between WT control and mutants that were secreted into medium. There was no significant difference in enzyme activity between WT and mutants. In this experiment, HEK293 cells cultured with equal cell numbers were transiently transfected with 500-ng constructs by effectane (Qiagen). Beginning at 48 h after transfection, cells were incubated for 15–20 h in complete serum-free DMEM. Media samples were collected and concentrated 20- to 30-fold by a 30K Ultrafree concentrating unit. Enzymatic activity assays followed the protocol described by Vindrola and Lindberg (1) using pGlu-Arg-Thr-Lys-Arg-MCA as the substrate. The ordinate represents activity relative to a standard sample prepared from a cultured medium pool. Values were also normalized according to the amount of secreted GFP fusion protein estimated by Western blotting.1. Vindrola, O. & Lindberg, I. (1993) Neuropeptides 25, 151–160.