Figure 6.

Cat F, but not Cat Z, degrades Iip10 from MHC class II–Iip10 complexes, generating MHC class II–CLIP. (A) N22 immunoprecipitates from metabolically radiolabeled Cat S^−/− mouse splenocytes treated with Con B (20 nM) were digested with either purified recombinant human Cat F or purified recombinant human Cat S at the indicated concentrations in assay buffer, pH 4.2, for 1 h at 37°C. The digested products were separated on 10–20% Tricine gel (Novex). (B) N22 immunoprecipitates were digested with 5 nM (as determined by cysteine protease active site titration; reference 10) of purified recombinant human Cat F–, S–, and B– or mouse Cat F– and Z–transfected 293 cell lysates and analyzed on a 10–20% Tricine gel. The Cat F and Z activity from cell lysate was examined with ¹²⁵I–JPM565 active site labeling. Densitometry analysis was used to quantitate the Iip10 digestion products of each Cat. CLIP-forming activities of recombinant Cat S and B are shown relative to Cat F (as 100%). Also, Cat Z–transfected 293 cell lysate CLIP-forming activity is shown relative to Cat F–transfected 293 cell lysate (as 100%).