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Fig. 4. Biosynthesis of cathepsin B, D and L in MPR-negative mouse embryonic fibroblasts. (A) MPR-negative MEFs lacking both M6/IGF2R and MPR46 were metabolically labeled with [³⁵S]methionine for 1 hour and then chased for up to 5 hours as described in Fig. 1. Procathepsin B (40-42 kDa), procathepsin D (46 kDa) and procathepsin L (37 kDa) were then sequentially immunoprecipitated from equivalent amounts of cell and medium extracts and analyzed by SDS-PAGE and fluorography. The mature forms of the proteinases were not detected in this experiment. The band labeled with an asterisk is nonspecific as described in Fig. 1. proCB, procathepsin B; proCD, procathepsin D; proCL, procathepsin L. (B) Microsomal extracts (15 µg total protein) of L-M(TK^-) cells (lane 1), Igf2r^-/- MEFs (lane 2) and MPR-negative MEFs (lane 3) were separated by SDS-PAGE and analyzed by immunoblotting with antibodies to cathepsin B, cathepsin D and cathepsin L. Band intensities determined by densitometry were used to compare the antigen contents of the samples. proCB, procathepsin B (42 kDa); scCB, mature cathepsin B (single-chain form; 30-32 kDa); scCD, mature cathepsin D (single-chain form; 43 kDa); tcCD, heavy-chain of mature cathepsin D (two-chain form; 29 kDa); proCL, procathepsin L (34-36 kDa); scCL, mature cathepsin L (single-chain form; 29 kDa); tcCL, heavy-chain of mature cathepsin L (two-chain form; 21 kDa).

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