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. 2016 Feb 5;291(14):7558–7570. doi: 10.1074/jbc.M115.701656

FIGURE 5.

FIGURE 5.

Selective binding of dextran-conjugated multimeric ligands on HEK 293T cells expressing CLL smIgs. Flow cytometry analysis of dextran-conjugated multimeric ligand binding to cells displaying CLL 014 smIg and other non-selecting control smIgs as controls on HEK 293T cells. The VH expression vectors were re-engineered by introducing a transmembrane domain at the C terminus of the CH3 constant region to express CLL BCR as smIgG. HEK 293T cells were transiently transfected with pIg γ-transmembrane and Igλ/κ plasmid pairs for CLL 014, CLL 068, CLL 169, and CLL 183 monoclonal Igs. The expression levels (bottom row) of the smIgs were determined by staining cells with anti-human Ig Fc-conjugated to allophycocyanin (anti-huIg Fc-APC) and analyzed on flow cytometry. The cells expressing smIgs were treated with bt-dext-KMS31 or bt-dext-KMS32 followed by staining with PE-conjugated streptavidin (sa-PE) before analysis by flow cytometry. Both of these multimeric ligands were selectively binding to cells only when they were expressing CLL 014 smIg. As shown, no significant staining was observed when cells were expressing non-selecting control CLL smIgs. bt-dex, biotinylated dextran.