ULBP2-BB4 binds to NK cells and CD138-expressing tumor cells simultaneously via NKG2D receptor and CD138 antigen. (A) ULBP2-BB4 was expressed in eucaryotic cell lines (293T and Cos) and purified with affinity chromatography using a 6× his tag. The eluted protein was separated on SDS-PAGE for Coomassie staining (left blot) and Western blotting with an anti-his–specific antibody (right blot). (B) Two CD138-positive cell lines were incubated with ULBP2-BB4 and labeled with an anti-his–FITC antibody for flow cytometry. The strong binding of ULBP2-BB4 (solid black line) could be competed after preincubation with a CD138-specific antibody (green line). (C) The binding of ULBP2-BB4 to RPMI-8226 and U-266 cells was visualized using soluble NKG2D receptor (NKG2DR) that was detected with an FITC-labeled anti-NKG2D antibody (dark line). No specific binding was seen using ULBP2-BB4 or soluble NKG2D receptor alone, both detected with the anti-NKG2D–FITC antibody (faint lines). (D) ELISA to measure the binding of ULBP2-BB4 (5 μg or 2.5 μg) to immobilized NKG2D receptor using an enzyme-linked anti-his antibody. An anti-NKG2D antibody (light gray bar) and the recombinant soluble single-chain BB4 that contained a 6× his tag (white bar) were used for the positive and the negative control, respectively. The mean OD_{492 nm} ± SD (n=3) is indicated.