Journal: The Journal of Biological Chemistry

Article Title: Interaction of Platelet Endothelial Cell Adhesion Molecule (PECAM) with α2,6-Sialylated Glycan Regulates Its Cell Surface Residency and Anti-apoptotic Role *

doi: 10.1074/jbc.M114.563585

Figure Lengend Snippet: Mouse PECAM mainly possesses sialylated biantennary N-glycans. A, a series of mutant Fc-PECAMs in which each potential N-glycosylation site was mutated were purified from overexpressing COS cells, subjected to SDS-PAGE, and visualized by silver-staining. B, immunopurified PECAM (arrowhead) from mouse lung tissues was verified by SDS-PAGE and silver staining. The de-N-glycosylated anti-PECAM IgG used for immunoaffinity chromatography is also shown (right lane). The only contaminant except for IgG, shown by the asterisk, was shown not to have any N-glycans because it was resistant to peptide N-glycosidase F treatment. C, PA-labeled N-glycans released from PECAM were separated by anion-exchange HPLC. The numbers shown indicate the eluted positions of standard N-glycans having 0, 1, 2, 3, and 4 sialic acid residues, respectively.

Article Snippet: Analysis of N-Acetyl Neuraminic acid (NeuAc) and N-Glycolyl Neuraminic Acid (NeuGc) Sialic acids were released from mouse tissue lysates (10 μg protein) or immunopurified PECAM (200 ng of protein) by acid hydrolysis and labeled with 1,2-diamino-4,5-methylenedioxybenzene using a sialic acid fluorescence labeling kit (TaKaRa).

Techniques: Mutagenesis, Purification, SDS Page, Silver Staining, Chromatography, Labeling