Intracellular Staining by Flow Cytometry
|Detection of Glypican 6 in HepG2 Human Cell Line by Flow Cytometry. HepG2 human hepatocellular carcinoma cell line was stained with Mouse Anti-Human Glypican 6 Monoclonal Antibody (Catalog # MAB2845, filled histogram) or isotype control antibody (Catalog # MAB002, open histogram), followed by Allophycocyanin-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # F0101B). To facilitate intracellular staining, cells were fixed with Flow Cytometry Fixation Buffer (Catalog # FC004) and permeabilized with Flow Cytometry Permeabilization/Wash Buffer I (Catalog # FC005). View our protocol for Staining Intracellular Molecules.|
The Glypicans (glypiated proteoglycans) are a small multigene family of GPI-linked heparan sulfate (HS) proteoglycans that likely play a key role in embryonic morphogenesis (1-4). There are currently six known mammalian Glypicans. They all share a common-sized protein core of 60‑70 kDa, an N-terminus which likely forms a compact globular domain, 14 conserved cysteines that form multiple intrachain disulfide bonds, and a number of C‑terminal N- and O-linked carbohydrate attachment sites. Based on exon organization and the location of O-linked glycosylation sites, at least two subfamilies of Glypicans are known, with one subfamily containing Glypicans 1, 2, 4 and 6, and another subfamily containing Glypicans 3 and 5 (3, 5). Human Glypican 6 (GPC-6) is synthesized as a 554 amino acid (aa) preproprecursor that contains a 23 aa signal sequence, a 505 aa mature region and a 26 aa C-terminal prosegment (5, 6). There are four consecutive Ser-Gly repeats that serve as a heparin sulfate attachment site. GPC-6 is reported to be as large as 110 kDa in size. This translates into approximately 50 kDa of proteoglycan (5). Human to mouse, there is 97% aa identity over the entire GPC-6 molecule. Cells known to express GPC-6 are adult ovary and embryonic vascular and visceral smooth muscle, plus mesenchyme (embryonic connective tissue) in multiple organs (1, 5, 6). The function of GPC-6 is essentially unknown. As a Glypican family member, it may facilitate heparin-binding growth factor signaling and polyamine uptake into expressing cells (7, 8). In this regard, it would appear that GPC-6 with its attendant HS is down‑regulated by triiodothyronine during cartilage maturation, thus limiting the availability of sites for FGF sequestration and activity (9).