Recombinant PC1-Gly593Arg (PC1-G593R) and PC1-Ala213del (PC1-A213Δ) were catalytically inactive. (a) Western blot analysis of CHO-K1 and βTC3 cells transfected with empty vector (C) or WT PC1, PC1-G593R, or PC1-A213Δ cDNA. Recombinant PC1 was detected with an anti-FLAG antibody. ΔCT, C-terminally truncated PC1. (b) Fluorogenic assay of PC1 proteolytic activity using anti-FLAG–immunopurified PC1 from lysates of βTC3 cells transfected as in a. Open circles, empty vector; filled diamonds, WT PC1 cDNA; open squares, PC1-G593R cDNA; open triangles, PC1-A213Δ cDNA. Activity was normalized for input of recombinant protein. (c) Co-immunoprecipitation of the propeptide of WT PC1 and PC1-A213Δ. Radiolabeled cell lysates (30-minute pulse, 1-hour chase) were immunoprecipitated with anti-FLAG antibody as in b. The lower part of the gel was exposed longer to visualize the propeptide.