Fig. 7: Biosynthesis of GPI is under regulation by ERAD in PIGS-KO cells. | Nature Communications

Fig. 7: Biosynthesis of GPI is under regulation by ERAD in PIGS-KO cells.

From: Cross-talks of glycosylphosphatidylinositol biosynthesis with glycosphingolipid biosynthesis and ER-associated degradation

Fig. 7

a Left: Flow cytometry analysis of ERAD gene knockout in PIGS-KO cells. Right: Quantitative data of MFI from three independent analyses (mean ± SD, n = 3). P values are from t test (unpaired and two-tailed) with comparisons to control (PIGS-KO). b Left: Flow cytometry analysis of PIGS-KO, PIGS-SLC35A2-DKO, PIGS-UBE2J1-DKO and PIGS-UBE2J1-SLC35A2-TKO HEK293 cells stained by T5 mAb. Right: Quantitative data of MFI from two independent experiments (mean ± SD, n = 2). See also Supplementary Fig. 7. c Western blotting of free GPI-GalNAc in ERAD-deficient cells. Lysates of PIGS-KO, PIGS-SLC35A2-DKO, PIGS-UBE2J1-DKO and PIGS-UBE2J1-SLC35A2-TKO cells were analyzed with T5 mAb. TfR, a loading control. d Top: PIGS-UBE2J1-DKO, PIGS-UBE2G2-DKO, PIGS-SYVN1-DKO and PIGS-DERL2-DKO HEK293 cells transiently expressing Vec or B3GALT4 were stained by 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 Vec control. e GPI biosynthesis. Left: Cells were metabolically labeled with [3H]mannose, GPI intermediates were extracted and analyzed by HPTLC. H5, GPI with one Man and one EtNP; H6, GPI with three Mans; H7, H7’, and H8, mature forms of GPI. Right: Quantitative data from two independent experiments (mean ± SD, n = 2). f Confirmation of KO of UBE2J1 or SYVN1 in SLC35A2-KO HEK293 cells by Western blot. GAPDH, a loading control. g GPI biosynthesis of ERAD-deficient SLC35A2-KO HEK293 cells. h Microarray of PIGS-KO and PIGS-UBE2J1-DKO HEK293 cells. Expression level changes of 36 genes of GPI pathway are shown. The UBE2J1 gene was significantly downregulated in the PIGS-UBE2J1-DKO cells, probably caused by nonsense-mediated mRNA decay. See also Supplementary Fig. 7b and Supplementary Data 4. i ERAD-deficiency enhances GPI biosynthesis. Genes validated are shown in light purple. Source data for (a), (b), (d), and (e) are provided as a Source Data file.

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