Fig. 7: O-glycosylation functions on SPACA7, INSL6, EQTN, and SPESP1.

The Flag-EGFP-tagged SPACA7 (a), INSL6 (c), EQTN (e), and SPESP1 (g) were transfected into CHO-K1 or CHO-ldlD cells. The VVA signals showing decreased levels of O-GalNAc glycan on proteins expressed in CHO-ldlD cells. (b) The role of O-glycosylation in protein stability was assessed. CHO cells were treated with 100 μg/mL CHX, and cell lysates were collected at various time points. The intensity of SPACA7 was normalized to that of GAPDH, and the percentage of remaining SPACA7 at different time points was calculated. d The role of O-glycosylation in protein maturation and cleavage was evaluated. The cell lysate and culture supernatant from the same batch of CHO cells were collected. The expression of pro-INSL6 and cleavage peptides were detected using anti-Flag antibody. f The role of O-glycosylation in protein-protein interaction was investigated. Co-Immunoprecipitation analysis was performed for EQTN and SNAP25. Flag-EGFP-tagged EQTN and His-EGFP-tagged SNAP25 were co-transfected into CHO-K1 or CHO-ldlD cells. Anti-Flag or anti-His antibody was used to detect the expression of EQTN or SNAP25. h The role of O-glycosylation in protein dimer formation was examined. Western blot analysis of cell lysates from the CHO cells transfected with SPESP1 under non-denatured or denatured conditions was detected by anti-Flag antibody. Data in (a–h) are representative of two or three independent experiments. VVA vicia villosa agglutinin, CHX cycloheximide, M marker, CBB Coomassie Brilliant Blue stain. Source data are provided as a Source Data file.