Active cathepsin B is localized to caveolae-enriched fractions of SUM149 3D cultures. Caveolae from SUM149 3D cultures were isolated by a detergent-based method, as described in Materials and Methods, that separates Triton X-100-soluble (TS; non-caveolae) from Triton X-100-insoluble (TI; caveolae) cellular fractions. (a) Equal-volume aliquots of each fraction were analyzed by SDS-PAGE and immunoblotted with antibodies against cathepsin B or caveolin-1. (b) TS and TI fractions were assayed for enzymatic activity against the synthetic cathepsin B substrate Z-Arg-Arg-NHMec. Cathepsin B activity in both TS and TI fractions was inhibited by 10 μM CA074; *, P < 0.01.