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

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Schematic of GSL pathway. Biosynthesis of Ganglio and Asiaio are shown. b Western blotting of 3HA tagged UGCG. Lysates of PIGS-UGCG-DKO cells stably expressing pLIB2-BSD-3HA (Vec), -UGCG-3HA (UGCG), and UGCG-D144A-3HA (D144A) were analyzed. GAPDH, a loading control. c Top: PIGS-UGCG-DKO cells stably expressing Vec, UGCG or D144A were stained with CTxB and T5 mAb. Bottom: 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 UGCG control. d Top: PIGS-B4GALT5-B4GALT6-TKO cells stably expressing Vec, B4GALT5 or D300A were stained with CTxB and T5 mAb. Bottom: 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 B4GALT5 control. e Western blotting of 3HA tagged B4GALT5. Lysates of PIGS-B4GALT5-B4GALT6-TKO cells stably expressing pLIB2-BSD-3HA (Vec), -B4GALT5-3HA (B4GALT5), and -B4GALT5-D300A-3HA (D300A) were analyzed. f Top: PIGS-ST3GAL5-DKO cells transiently expressing Vec or mouse ST3GAL5 and PIGS-B4GALNT1-DKO cells transiently expressing Vec or human B4GALNT1 were stained with CTxB, T5 mAb, and anti-LacCer mAb. Bottom: Quantitative data of MFI from two independent experiments (mean ± SD, n = 2). g Top: PIGS-A4GALT-DKO cells stably expressing Vec or human A4GALT and PIGS-B3GNT5-DKO cells stably expressing Vec or human B3GNT5 were stained with CTxB, T5 mAb, and anti-LacCer mAb. Bottom: Quantitative data of MFI from two independent experiments (mean ± SD, n = 2). See also Supplementary Fig. 5c and d. h Quantitative data of high-performance thin-layer chromatography profile of [¹⁴C] galactose-labeled GSLs from HEK293 cells. Data from two independent experiments (mean ± SD, n = 2). See also Supplementary Fig. 5e. i Top: PIGS-B4GALT5-ST3GAL5-TKO cells transiently expressing Vec or mouse ST3GAL5 and PIGS-B4GALT5-DKO cells were stained with CTxB, T5 mAb, and anti-LacCer mAb. Bottom: Quantitative data of MFI from four independent experiments (mean ± SD, n = 4). P values are from one-way ANOVA followed by Dunnett’s test for multiple comparisons to Vec. Source data for (c, d), (f–h), and (i) are provided as a Source Data file.