Intracellular Staining by Flow Cytometry
Detection of Phospho-Axl (Y779) in HeLa Human Cell Line by Flow Cytometry.HeLa human cervical epithelial carcinoma cell line either (A) untreated or (B) treated with 100 nM Pervanadate for 10 minutes was stained with Mouse Anti-Human Phospho-Axl (Y779) APC‑conjugated Monoclonal Antibody (Catalog # IC6965A) and Mouse Anti-Human Axl PE‑conjugated Monoclonal Antibody (Catalog # FAB154P). Quadrant markers were set based on control antibody staining (Catalog # IC002A). 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.
Axl (Ufo, Ark), Dtk (Sky, Tyro3, Rse, Brt), and Mer (human and mouse homologues of chicken c-Eyk) constitute a subfamily of the receptor tyrosine kinases (1, 2). The extracellular domains of these proteins contain two Ig-like motifs and two fibronectin type III motifs. This characteristic topology is also found in neural cell adhesion molecules and in receptor tyrosine phosphatases. The human Axl cDNA encodes an 887 amino acid (aa) precursor that includes an 18 aa signal sequence, a 426 aa extracellular domain, a 21 aa transmembrane segment, and a 422 aa cytoplasmic domain. The extracellular domains of human and mouse Axl share 81% aa sequence identity. A short alternatively spliced form of human Axl is distinguished by a 9 aa deletion in the extracellular juxtamembrane region. These receptors bind the vitamin K-dependent protein growth arrest specific gene 6 (Gas6) which is structurally related to the anticoagulation factor Protein S. Binding of Gas6 induces receptor autophosphorylation and downstream signaling pathways that can lead to cell proliferation, migration, or the prevention of apoptosis (3). This family of tyrosine kinase receptors is involved in hematopoiesis, embryonic development, tumorigenesis, and regulation of testicular functions. Phosphorylation of Tyrosine 779 provides a docking site for p85 subunits of PI 3-Kinase (4).