Core 1 O-glycans protect PDPN from proteolytic degradation. (A) Stable PDPN-eGFP transfected WT or C1galt1^−/− ECs were incubated in buffer (control), 0.05% trypsin (with or without 5 mM benzamidine), 100 μg/ml elastase (with or without 2 mM PMSF), 10 μg/ml calpain-2 (with 2 mM Ca⁺⁺, with or without 5 mM EDTA), or 2 μg/ml activated MMP-2/MMP-9 (with or without 100 μM GM6001), and then stained with anti-PDPN antibody for flow cytometry. The bar graph indicates the percentage of MFI of cell surface PDPN levels against that of control. The data represent the mean ± SD from 3 to 5 experiments. ***P < .001. (B) Stable WT or C1galt1^−/− ECs were treated with or without trypsin, elastase, calpain-2, or activated MMP-2/MMP-9, and then lysed. All lysates were analyzed by immunoblot using anti-PDPN mAb (8.1.1) or rabbit anti-GFP Ab. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. (C) Potential O-glycosylation sites (black dots) and predicted cleavage sites of PDPN by trypsin (yellow), elastase (green), calpain-2 (red), or MMP-2/MMP-9 (purple) in the extracellular domain of PDPN.