BJAB transfectants have similar levels of expression of Fas receptor, and BJABK1 cells recruit fewer subunits of Fas, FADD, and procaspase 8 to the DISC after stimulation with an anti-Fas antibody. (A) Expression of Fas on the surface of BJAB transfectants. Stably transfected BJAB cells were stained with the anti–mouse IgG–FITC antibody, and expression of Fas was detected by flow cytometry. The levels of Fas expression were the same in BJABK1, BJABK1m, and BJABXS cells. (B) Suppression of the cleavage of procaspase 8 by expression of K1. BJAB transfectants were evaluated for their level of procaspase 8 cleavage with the anti-Fas antibody CH-11. After 30 or 60 minutes of incubation with CH-11, BJABK1 cells contained low levels of procaspase 8 cleavage products (indicated by arrowheads). Recombinant caspase 8 was used as a positive control. Similar results were obtained in 3 independent experiments. (C) Caspase 8 activity in BJAB transfectants. BJAB transfectants were incubated with CH-11; at periodic intervals, extracts were removed and incubated with the caspase 8 substrate IETD-AFC at 37°C for 2 hours, and caspase 8 activity was analyzed by using a fluorescence reader. BJABK1 cells had a significantly lower level of caspase 8 activity than BJABK1m and BJABXS cells (P < .005). Caspase 8 activity was completely inhibited by treatment with the caspase 8 inhibitor z-IETD-fmk. Error bars represent SD. Similar results were obtained in 2 independent experiments. (D) BJABK1, BJABK1m, and BJABXS cells were treated with 1 μg/mL CH-11 for the indicated times. Cell extracts were subjected to immunoprecipitation (IP) with CH-11 and immunoblotting (IB) with antibodies targeting procaspase 8, FADD, and FLIP. All of the transfectants recruited these DISC subunits as indicated by the bands shown. The intensity of the p43/p41 and FADD bands was lower in K1 compared with vector-transfected cells. Fas and cFLIP levels remained unchanged. Similar results were obtained in 3 independent experiments.