|Detection of Human Phospho-M-CSF R/CD115 (Y723) by Western Blot. Western blot shows lysates of M‑CSF R/CD115‑transfected NS0 mouse myeloma cell line untreated (-) or treated (+) with 100 ng/mL Recombinant Human M‑CSF/CD115 (Catalog # 216-MC) for 10 minutes. PVDF membrane was probed with 0.5 µg/mL of Rabbit Anti-Human Phospho-M-CSF R/CD115 (Y723) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3268), followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). A specific band was detected for Phospho-M-CSF R/CD115 (Y723) at approximately 175 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.|
M-CSF R/CD115 in Human PBMCs.|
M-CSF R/CD115 phosphorylated at Y723 was detected in immersion fixed human peripheral blood mononuclear cells (PBMCs) using Rabbit Anti-Human
Phospho-M-CSF R/CD115 (Y723) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3268) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Rabbit IgG Secondary Antibody (red; Catalog # NL004) and counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Non-adherent Cells.
M-CSF R, the product of the c-fms proto-oncogene, is a member of the type III subfamily of receptor tyrosine kinases that also includes receptors for SCF and PDGF. These receptors each contain five immunoglobulin-like domains in their extracellular domain (ECD) and a split kinase domain in their intracellular region (1‑4). M-CSF R is expressed primarily on cells of the monocyte/macrophage lineage, dendritic cells, stem cells and in the developing placenta (1). Human M-CSF R cDNA encodes a 972 amino acid (aa) type I membrane protein with a 19 aa signal peptide, a 493 aa extracellular region containing the ligand-binding domain, a 25 aa transmembrane domain and a 435 aa cytoplasmic domain. The human M-CSF R ECD shares 60%, 64%, 72%, 75%, 75% and 76% aa identity with mouse, rat, bovine, canine, feline and equine M-CSF R, respectively. Activators of protein kinase C induce TACE/ADAM17 cleavage of the M-CSF R, releasing the functional
ligand-binding extracellular domain (5). M-CSF binding induces receptor homodimerization, resulting in transphosphorylation of specific cytoplasmic tyrosine residues and signal transduction (6). The intracellular domain of activated M-CSF R binds more than 150 proteins that affect cell proliferation, survival, differentiation and cytoskeletal reorganization. Among these, PI3Kinase, P42/44 ERK and c-Cbl are key transducers of M-CSF R signals (3, 4). M-CSF R engagement is continuously required for macrophage survival and regulates lineage decisions and maturation of monocytes, macrophages, osteoclasts and DC (3, 4). M-CSF R and integrin alpha v beta 3 share signaling pathways during osteoclastogenesis and deletion of either causes osteopetrosis (7, 8). In the brain, microglia expressing increased M-CSF R are concentrated with Alzheimers a beta peptide, but their role in pathogenesis is unclear (9, 10).