Sensitivity of endomucin to O-sialoglycoprotein endopeptidase. (A) bEND.3 cells were treated with O-sialoglycoprotein endopeptidase from Pasteurella haemolytica for 30 minutes (dotted line) or left untreated (thick line) and analyzed directly by flow cytometry as described for Fig 5A. The profiles show the binding of rat MoAb V.5C7 (endomucin), MoAb 390 (CD31), MoAb SM8 (CD59), and MoAb III.3A7 (Sca-2). Negative control staining was analyzed with MoAb MRC OX86 (thin line). (B) bEND.3 cells were surface biotinylated and subjected to immunoprecipitation with the antiendomucin antibody MoAb V.5C7 (lanes 1 through 3) or the anti–PECAM-1 antibody EA-3 (lanes 4 through 6). Immunoprecipitated antigens were either mock-treated (lanes 1 and 4), treated with O-sialoglycoprotein endopeptidase (lanes 2 and 5), or treated with neuraminidase (lanes 3 and 6). After the enzyme treatment, proteins were electrophoresed on a 10% polyacrylamide gel, transferred to nitrocellulose, and detected with peroxidase-conjugated streptavidin followed by enhanced chemoluminescence. Molecular mass markers (in kilodaltons) are indicated on the left.