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. 2021 May 11;12:2672. doi: 10.1038/s41467-021-22618-x

Fig. 2. B7H3 is N-glycosylated at NXT motif sites in TNBC cells.

Fig. 2

a Cell lysates from MDA-MB-231 and HCC1806 cells were treated with PNGase F, Endo H, and O-glycanase for 1 h at 37 °C in vitro. b Cell lysates from six TNBC tumors were treated with PNGase F for 1 h at 37 °C in vitro. c MDA-MB-231 and HCC1806 cells were treated with TM (2.5 μg/ml), Thiamet G (50 μM), and PUGNAc (100 μM) for 24 h. d Schematic diagram of human B7H3-8NQ mutants used in this study (upper). Effect of human B7H3 knockout in MDA-MB-231 and HCC1806 cells using CRISPR–Cas9 technology. Then the B7H3KO cells were stably rescued with B7H3-WT-Flag and B7H3-8NQ-Flag cDNA (bottom). e Schematic diagram of mouse B7H3-4NQ mutants used in this study(upper). Effect of mouse B7H3 knockout in 4T1 cells using CRISPR–Cas9 technology. 4T1-B7H3KO cells were stably rescued with B7H3-WT-Flag and B7H3-4NQ-Flag cDNA (bottom). Black closed circle, non-specific band. f Cell lysates from the indicated cell lines were treated with PNGase F, Endo H, and O-glycanase for 1 h at 37 °C in vitro. g The indicated cell lines were treated with N-linked glycosylation inhibitors TM (2.5 μg/ml), SW (5 μg/ml), and DMJ (10 μg/ml), or O-linked glycosylation inhibitors Thiamet G (50 μM) and PUGNAc (100 μM) for 24 h. SW, swainsonine; DMJ, deoxymannojirimycin. h Nano LC-MS/MS of the N-glycans on positions N91, N309, N104, N322, N189, N407, and N215 and N433 of purified human B7H3 protein from wild-type B7H3 re-expressed MDA-MB-231-B7H3KO cells. All data are representative of three independent experiments. Red closed circle, glycosylated B7H3; blue star, non-glycosylated B7H3.