Fig. 6.

SapB binds and loads short, but not long, CD1 lipid antigens. (A) GD1b (8 μg) was passed through cellulose nickel columns and treated with 0.2 M imidazole buffer. Buffer alone (Left), column eluate (Center), and organic wash eluate (Right; 2:1 chloroform:methanol wash), were spotted directly on a TLC silica plate. Salt was removed by running plate with deionized water and were subsequently sprayed with α-naphthol to detect glycolipids (B) 6x-Histidine–tagged SapB (5 μg) was incubated on its own or with the ganglioside GD1b (4 μg), C₃₂ GMM (8 μg), or C₈₀ GMM (8 μg) at pH 5 for 16 h. Following incubation, samples were passed through columns packed with nickel chelating cellulose. After extensive washing, columns were treated with 0.2 M imidazole, and eluates were spotted directly on a TLC silica plate. Salt was removed by running the plate with deionized water. Plates were allowed to dry in a vacuum. Dried plates were sprayed with α-naphthol to detect glycolipids. Plates were then charred to visualize signal. (C) Sonicated C₃₂ GMM (1 μg) or C₈₀ GMM (1 μg) was incubated with saposin proteins (5.4 μM) as indicated and CD1b-Fc fusion protein in sodium citrate buffer, pH 5.0. Fusion protein samples were immobilized on protein G-coated plates. After washing with PBS solution, CD1b-restricted T cells (LDN5 T cells) were added and incubated in complete media for 20 h. IFN-γ released by activated T cells was measured by ELISA.