Fig. 4. Activity of procathepsin L and cathepsin L against a peptide analog and mNC1. (A) SFCM from EOMA cell cultures was adjusted to the pH indicated, probed with the active site-directed reagent [¹²⁵I]LHVS-PhOH for 1 h at 37°C and analyzed by autoradiography. (B) Coomassie Blue staining and [¹²⁵I]LHVS-PhOH labeling of the purified enzyme fraction. (C) A 100 ng aliquot of recombinant mNC1 was incubated with 10 ng of the purified enzyme fraction for increasing time periods at pH 5.5 and 7.4. The samples were then probed by Western blotting with an anti-flag antibody (upper panel) as well as an antibody against murine procathepsin L (lower panel). (D) A 10 ng aliquot of procathepsin L was first converted into mature cathepsin L by a 30 min incubation at pH 5.5 and then incubated with 100 ng of recombinant mNC1 at pH 5.5 and 6.8 and analyzed as in (B) (Cat L = cathepsin L).