DR5Δ blocks IFN-induced apoptosis.

U266 cells were transfected with pcDNA3-DR5Δ (residues 1-268). DR5Δ (also called TRAIL-R2) lacks the death domain and has been shown to function as a dominant-negative molecule inactivating the function of the endogenous DR5 (alsoTRAIL-R2).35 DR5Δ also contains a FLAG epitope-tag that facilitates examination of its expression levels (A). Parental and DR5Δ-FLAG–containing U266 cells were treated with Apo2L (100 ng/mL), IFN-α, and -β (200 U/mL), and apoptosis was determined at 72 hours by Hoechst 33258 staining and expressed as the percentage of the total cells. At least 200 cells were counted for each sample. Expression of DR5Δ was determined by Western blotting using an anti-FLAG antibody and β-actin as a protein loading control (inset). In a parallel experiment (B), the cells were also lysed and subjected to Western blotting for caspase 8 and Bcl-2, upper and lower panel, respectively.