Fig. 1.

Confirmation of Lc₃Cer synthase activity in B3gnt5^−/− mice and MS analysis of the glycans derived from GSLs of mouse B cells. (A) Homogenates (100 μg of protein) of mouse tissues were used to assay Lc₃Cer synthase activity. The products were separated on a high-performance twin-layer chromatography (HPTLC) plate with a solvent system of chloroform/methanol/0.2% CaCl₂ (60:35:8 vol/vol/vol). The intensities of the radioactive bands were measured with a FLA3000 Image Analyzer (Fujifilm, Tokyo). HL-60, which has Lc₃Cer synthase activity, and Jurkat, which does not, were used as positive and negative controls, respectively. Lc₃Cer synthase assays were performed on two TLC plates, with positive and negative control on each plate. This figure was prepared from the results of two TLC plates to demonstrate Lc₃Cer synthase activity in cerebellum, spleen, and colon between WT and B3gnt5^−/− mice. (B) MS spectra of the glycans derived from GSLs of mouse B cells. Arrows indicate glycan signals that are absent from GSLs of B3gnt5^−/− B cells. Arrowheads indicate glycan signals that are decreased in GSLs of B3gnt5^−/− B cells. Table S1 summarizes the results of the assigned GSL-derived glycan structures of mouse B cells. Detailed methods for MS of glycans derived from GSLs are described in Fig. S2.