Fig. 2. Stable introduction of FAP-1 renders Capan-1 cells resistant to CD95-induced apoptosis. (A) Immunoblot analysis of FAP-1 and CD95 in Capan-1 transfectants. Data shown are derived from a single exposure of two different blots of which irrelevant lanes have been removed for clarity of presentation. 40 µg of protein was loaded for Capan-1, 8 µg for Panc89. An antibody to ß-actin was used as a loading control. (B) Relative sensitivity of FAP-1 transfectants to CD95-induced apoptosis (24 hour treatment with agonistic anti-CD95 antibody CH11, 100 ng/ml) measured with the JAM assay. Data for clone 2 are given as % viability (mean ± s.d.) relative to untreated cells set at 100%; equivalent data for clones 4, 12 and 40 are presented as the ratio of viability (%) of each FAP-1 transfectant over vector control. (C) Immunoblot analysis of PARP cleavage in Capan-1 (parental), vector-transfected control (vector) and Capan-1-FAP-1 clone 2 (clone 2) cells stimulated for 24 hours with CH11 (100 ng/ml). The appearance of the 85 kDa PARP cleavage product is indicative of caspase-3 activity. (D) Immunoblot analysis of caspase-8 cleavage after anti-CD95 (Ab) or TRAIL (Tr) stimulation. Capan-1 cells were left untreated (Co) or were treated for 24 hours with either CH11 antibody (Ab, 100 ng/ml) or TRAIL (Tr, 100 ng/ml). The degree of caspase-8 (casp 8) cleavage corresponds to a decrease in the procaspase-8-specific signal and should be estimated relative to the intensities of the ß-actin bands.