Fig. 6

B3GNT5 was N-glycosylated, and glycosylation of B3GNT5 stabilized its protein. A Cell lysates from HEK293T cells expressing B3GNT5-WT-Flag were treated with PNGase F or O-glycosidase for 2 h at 37 °C in vitro. Band shift was detected by Western blotting. B Schematic diagram of each asparagine mutated site and human B3GNT5-4NQ mutant used in this study. C Expression pattern of each asparagine mutated site was examined by Western blotting in HEK293T cells. Red closed circle, glycosylated B3GNT5 (B3GNT5-WT); blue star, non-glycosylated B3GNT5 (B3GNT5-4NQ). D Cell lysates from HEK293T with B3GNT5-4NQ expression was treated with PNGase F or O-glycosidase for 2 h at 37 °C in vitro. Band shift was detected by Western blotting. E HEK293T cells with B3GNT5-WT or B3GNT5-4NQ expression were treated with CHX (20 µM) for indicated intervals. F HEK293T cells with B3GNT5-WT or B3GNT5-4NQ expression were treated with MG132 (20 µM) for 8 h. G HEK293T cells with B3GNT5-WT or B3GNT5-4NQ expression were treated with tunicamycin (TM, 2.5 µg/mL) for 24 h. B3GNT5 protein level was detected using Western blotting. H HEK293T cells with stable empty vector, wild-type B3GNT5 or different mutants were used for measuring surface level of SSEA-1. Data are shown as a percentage of control cell line. Data are shown as mean ± SD based on three independent experiments. I, J Expression efficiency of B3GNT5-WT-Flag and B3GNT5-4NQ-Flag was measured in MDA-MB231 KO (I) and BT549 KO cells (J). K, L Surface expression of SSEA-1 was detected using flow cytometry in indicated cell lines. M, N Soft-agar assay (M) and mammosphere assay (N) were performed using empty vector, B3GNT5-WT-Flag or B3GNT5-4NQ-Flag re-expressing MDA-MB231 KO cells and BT549 KO cells. Data are presented as a percentage of control cell lines as in (H)