Skip to main content
. 2020 Feb 13;11:860. doi: 10.1038/s41467-020-14678-2

Fig. 3. B3GALT4 transfers Gal to both GM2 and GPI-GalNAc.

Fig. 3

a B3GALT4, which is required for transfer Gal to GA2 and GM2 to generate GA1 and GM1a, is the candidate for GPI-Gal-T. b Left: PIGS-KO (top) and PIGS-B3GALT4-DKO HEK293 cells stably expressing pME-Puro (Vec) or pME-Puro-hB3GALT4-3FLAG (B3GALT4) were stained with CTxB and T5 mAb. Right: Quantitative data of MFI from three independent experiments (mean ± SD, n = 3). P values are from t test (unpaired and two-tailed) with comparisons to vector control. See also Supplementary Fig. 2a. c Representative images of cells labeled with CTxB on ice, fixed, and stained with T5 mAb. Scale bar, 10 μm. d Western blotting of free GPI-GalNAc. Lysates of PIGS-KO and PIGS-B3GALT4-DKO HEK293 cells stably expressing Vec or B3GALT4 were analyzed by Western blotting. TfR, a loading control. e Expected structures of GPI-bearing C-terminal peptides from HFGF-CD59 that was released from HEK293 cells by PI-PLC and digested with trypsin. The C-terminal peptides linked to GPI of glycoforms 1–8 are shown. f Quantitative data of LC-ESI-MS/MS analysis. Percentage of total intensity was calculated from the peak areas. See also Supplementary Fig. 2b–d and 3, and Supplementary Table 2. g Expression of catalytic mutant of B3GALT4 confirmed by western blotting. TfR, a loading control. h Left: Flow cytometry analyses of PIGS-B3GALT4-DKO cells transiently expressing pME-hB3GALT4-3HA (WT), -hB3GALT4-D175A-3HA (D175A), -hB3GALT4-D176A-3HA (D176A), -hB3GALT4-D177A-3HA (D177A), and -hB3GALT4-D291A-3HA (D291A). Right: Quantitative data of MFI from at least four independent experiments (mean ± SD, n ≥ 4). P values are from one-way ANOVA followed by Dunnett’s test for multiple comparisons to control (WT). See also Supplementary Fig. 4b–e. Source data for (b) and (h) are provided as a Source Data file.