CD44 is a type 1 transmembrane glycoprotein also known as Phagocytic Glycoprotein 1 (pgp 1) and HCAM. CD44 is the receptor for hyaluronate and exists as a large number of different isoforms due to alternative RNA splicing. The major isoform expressed on lymphocytes, myeloid cells, and erythrocytes is a glycosylated type 1 transmembrane protein. Other isoforms contain glycosaminoglycans and are expressed on hematopoietic and non hematopoietic cells. CD44 is involved in adhesion of leukocytes to endothelial cells, stromal cells, and the extracellular matrix.
Function:
Receptor for hyaluronic acid (HA). Mediates cell-cell and cell-matrix interactions through its affinity for HA, and possibly also through its affinity for other ligands such as osteopontin, collagens, and matrix metalloproteinases (MMPs). Adhesion with HA plays an important role in cell migration, tumor growth and progression. Also involved in lymphocyte activation, recirculation and homing, and in hematopoiesis. Altered expression or dysfunction causes numerous pathogenic phenotypes. Great protein heterogeneity due to numerous alternative splicing and post-translational modification events.
Subcellular Location:
Membrane.
Tissue Specificity:
Isoform 10 (epithelial isoform) is expressed by cells of epithelium and highly expressed by carcinomas. Expression is repressed in neuroblastoma cells.
Post-translational modifications:
Proteolytically cleaved in the extracellular matrix by specific proteinases (possibly MMPs) in several cell lines and tumors.
N-glycosylated.
O-glycosylated; contains more-or-less-sulfated chondroitin sulfate glycans, whose number may affect the accessibility of specific proteinases to their cleavage site(s).
Phosphorylated; activation of PKC results in the dephosphorylation of Ser-706 (constitutive phosphorylation site), and the phosphorylation of Ser-672.
Similarity:
Contains 1 Link domain.
SWISS:
P16070
Gene ID:
960
Database links:
Entrez Gene: 100034221 Horse
Entrez Gene: 960 Human
Entrez Gene: 12505 Mouse
Omim: 107269 Human
SwissProt: Q05078 Horse
SwissProt: P16070 Human
SwissProt: P15379 Mouse
Unigene: 502328 Human
Unigene: 423621 Mouse
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Sample:
Lane 1: Huvec (Human) Cell Lysate at 30 ug
Lane 2: A549 (Human) Cell Lysate at 30 ug
Primary: Anti-CD44v5 (SL2782R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 110-36 kD
Observed band size: 110-36 kD
Tissue/cell: human laryngo carcinoma; 4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,SLC0005) at 37℃ for 20 min;
Incubation: Anti-CD44v5 Polyclonal Antibody, Unconjugated(SL2782R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) staining
Blank control: HELA.
Primary Antibody (green line): Rabbit Anti-CD44v5 antibody (SL2782R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-AF647
Dilution: 1μg /test.
Protocol
The cells were incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.
Blank control(blue): U251 (fixed with Ice-cold 70% ethanol overnight at 4 °C).
Primary Antibody:Rabbit Anti-CD44v5 antibody (SL2782R,Green); Dilution: 1μg in 100 μL 1X PBS containing 0.5% BSA;
Isotype Control Antibody: Rabbit IgG(orange), used under the same conditions;
Secondary Antibody: Goat anti-rabbit IgG-FITC(white blue), Dilution: 1:200 in 1 X PBS containing 0.5% BSA.
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