Perforin Activity Modulation by Cathepsin B

Human purified perforin (Enzo Life) permabilizing activity was determined by 1 h incubation of 1 × 10⁶ Jurkat cells with 125 ng of perforin following manufacturer's recommended conditions. Perforin was pretreated or not for 2 h at 37 °C with 500 ng ml⁻¹ purified human liver cathepsin B (CatB, Merck-Millipore), or CatB that was previously co-incubated with 10 μM CA074 (Merck-Millipore) for 30 min at 37 °C. Cell permeabilization was measured in the presence of 25 μg ml⁻¹ PI by FACS analysis. In some experiments, lytic granule lysates were pretreated or not with melanoma cell vesicular fraction lysates for 2 h at 37 °C. Melanoma cell vesicular fraction lysates were previously co-incubated or not with 10 μM CA074 (Merck-Millipore) for 30 min at 37 °C before being incubated with lytic granule lysates. Intracellular vesicles isolation from melanoma cells was performed following a published protocol57 (link), that we previously employed for lytic granule isolation with minor modifications (see below).