Intracellular Staining by Flow Cytometry
Detection of Collagen XXIII alpha 1 in NS0 Mouse Cell Line Transfected with Human Collagen XXIII alpha 1 by Flow Cytometry. NS0 mouse myeloma cell line transfected with human Collagen XXIII alpha 1 was stained with Sheep Anti-Human Collagen XXIII alpha 1 Alexa Fluor® 647‑conjugated Antigen Affinity-purified Polyclonal Antibody |
(Catalog # IC4165R, filled histogram) or isotype control antibody (Catalog # IC016R, open histogram). To facilitate intracellular staining, cells were fixed and permeabilized with FlowX FoxP3 Fixation & Permeabilization Buffer Kit (Catalog # FC012). View our protocol for Staining Intracellular Molecules.
Collagen XXIII alpha 1 (sometimes abbreviated COL23A1) is a ~75 kDa type II transmembrane nonfibrillar collagen that is a member of the collagenous transmembrane protein superfamily (1, 2). This family also includes collagens XIII, XVII, XXV and non‑collagens with triple‑helical regions such as ectodysplasin A, class A macrophage scavenger receptors, and MARCO (2, 3). The human Collagen XXIII mRNA encodes a 540 amino acid (aa) protein containing a 34 aa N‑terminal cytoplasmic domain, a 21 aa transmembrane (TM) domain and a 485 aa extracellular domain (ECD). The ECD contains a coiled‑coil consensus sequence to aid homotrimerization (aa 64‑69), a furin cleavage site (aa 105‑110), a pair of cysteines thought to form intermolecular disulfides (aa 106 and 108), and three collagen domains (1, 3‑5). The C‑terminal 20 aa, including cysteines at aa 525 and 537 of Collagen XXIII, is conserved among TM collagen proteins. Proteolytic cleavage, occurrs mainly in the Golgi, and allows the Collagen XXIII ectodomain to be secreted as a soluble trimer of ~60 kDa subunits (1, 6). Cell surface cleavage can also occur, but is slow, presumably due to the presence of Collagen XXIII in lipid raft membrane domains (6). The protein database includes three variants of 537, 316 and 309 aa with various portions missing or substituted; all appear to lack TM segments (7). The human Collagen XXIII ECD shares 92%, 93%, and 91% aa sequence identity with mouse, rat, and canine Collagen XXIII, respectively. Collagen XXIII is concentrated at sites of cell contact in epithelia, and is now known to bind to Integrin alpha 2 beta 1 on the surface of stratum basale keratinocytes (2, 5, 8).