Design and biochemical characterization of the MUC16-targeted TRAIL trimer TR3. A, Schematic representation of the proteins used in this study. Soluble mesothelin (Meso) containing an N-terminal FLAG tag (not shown), the parental TRAIL drug platform TR3 (center) and the MUC16-targeted mesothelin-TR3 fusion protein (Meso-TR3) were produced by transient transfection of HEK293T cells. N, amino-terminus; C, carboxyl-terminus. B, Western blot analysis (reducing conditions) documents the molecular weights of TR3 (≈60 kDa, lane 2) and Meso-TR3 (≈100 kDa, lane 3) using anti-TRAIL pAb. Supernatant from mock-transfected HEK293T cells served as a negative control (lane 1).