Effects of neutralizing antibodies to TRAIL receptors on TRAIL-induced apoptosis and expression of TRAIL receptors on the cell surface in PC3-MM2 and KM12L4A cells. (A) PC3-MM2 and KM12L4A cells were pretreated with the anti-DR4 or anti-DR5 antibodies (0.5 μg/ml) for 3 h and then PC3-MM2 and KM12L4A cells were treated with the indicated doses of TRAIL for 6 and 8 h, respectively. Goat IgG was used as the control isotype antibody. Apoptosis was detected by annexin V binding assay. Each value represents the mean ± SE of triplicate determinants. ***p < 0.001. (B) PC3-MM2 and KM12L4A cells were treated with 2.5 ng/ml TRAIL for 3 h. Thereafter, cells were stained with control mouse IgG or anti-DR4/DR5 antibody (1:100), and subsequently labeled with FITC-conjugated secondary antibodies (1:200) to determine the surface expression of DR4/DR5. The cell surface expression was measured by a flow cytometer. Shaded and unshaded peaks correspond to control and specific stainings, respectively.