FIG. 4.

Biological activity of ACRP:FasL, ACRP:CD40L, and ACRP:Tweak. (A) FasL-sensitive 293T-6 cells transfected with a Flag-JNK expression plasmid were treated for the indicated period of time with FasL or ACRP:FasL in the presence or absence of cross-linking antibody. Cell lysates were analyzed by Western blotting using an anti-phospho-JNK antibody. Equal expression levels of JNK were confirmed by reprobing the membrane with anti-Flag antibody. (B) Jurkat cells were treated with FasL and ACRP:FasL as described in the Fig. 3B legend, except that the caspase inhibitor Z-VAD-fmk was added to reveal caspase-independent cell death. (C) Purified murine splenic B cells were incubated for 48 h with the indicated concentration of murine CD40L (squares) or ACRP:CD40L (circles) in the presence (filled symbols) or absence (open symbols) of cross-linking anti-Flag M2 antibody. Proliferation was measured by [³H]thymidine incorporation. Controls include LPS (triangles) and heat-inactivated ACRP:CD40L (diamonds). Figure shows the mean plus or minus standard deviation of triplicates. (D) The Kym-1 rhabdomyosarcoma cell line was treated with Tweak and ACRP:Tweak, as indicated. Cell viability was monitored using PMS/MTS reagents.