Detection of LAP (TGF-beta 1) in Mouse CD4+ iTreg cells by Flow Cytometry. Mouse splenocytes treated with 10 μg/mL Anti-CD3, 5 μg/mL Anti-CD28, 10 μg/mL Recombinant Human TGF‑ beta 1 (Catalog # 240-B), and 20 μg/mL Recombinant Mouse IL‑2 (Catalog # 402-ML) for 24 hours to generate induced T regulatory cells (iTregs) were stained with Rat Anti-Mouse CD25/IL-2 R alpha APC‑conjugated Monoclonal Antibody (Catalog # FAB2438A) and either (A) Rat Anti-Mouse LAP (TGF-beta 1) PE‑conjugated Monoclonal Antibody (Catalog # FAB8118P) or (B) Rat IgG2A Phycoerythrin Isotype Control (Catalog # IC006P). Cells were gated on CD4+ iTregs. View our protocol for Staining Membrane-associated Proteins.
Preparation and Storage
The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage
Protect from light. Do not freeze.
12 months from date of receipt, 2 to 8 °C as supplied.
Background: LAP (TGF-beta 1)
TGF-beta 1 (Transforming Growth Factor beta 1) is one of three closely related mammalian members of the large TGF-beta superfamily that share a characteristic cystine knot structure (1‑7). TGF-beta 1, -2 and -3 are highly pleiotropic cytokines that are proposed to act as cellular switches that regulate processes such as immune function, proliferation and epithelial-mesenchymal transition (1‑4). Each TGF-beta isoform has some non‑redundant functions; for TGF-beta 1, mice with targeted deletion show defects in hematopoiesis and endothelial differentiation, and die of overwhelming inflammation (2). Human TGF‑ beta 1 cDNA encodes a 390 amino acid (aa) precursor that contains a 29 aa signal peptide and a 361 aa proprotein (8). A furin‑like convertase processes the proprotein to generate an N‑terminal 249 aa Latency‑Associated Peptide (LAP) and a C‑terminal 112 aa mature TGF‑ beta 1 (8, 9). Disulfide‑linked homodimers of LAP and TGF‑ beta 1 remain non‑covalently associated after secretion, forming the small latent TGF‑ beta 1 complex (8‑10). Covalent linkage of LAP to one of three latent TGF‑ beta binding proteins (LTBPs) creates a large latent complex that may interact with the extracellular matrix (9, 10). TGF‑ beta is activated from latency by pathways that include actions of the protease plasmin, matrix metalloproteases, thrombospondin 1 and a subset of integrins (10). Mature human TGF‑ beta 1 shares 100% aa identity with pig, dog and cow TGF‑ beta 1, and 99% aa identity with mouse, rat and horse TGF‑ beta 1. It demonstrates cross‑species activity (1). TGF‑ beta 1 signaling begins with high‑affinity binding to a type II ser/thr kinase receptor termed TGF‑ beta RII. This receptor then phosphorylates and activates a second ser/thr kinase receptor, TGF‑ beta RI, also known as Activin Receptor‑Like Kinase 5(ALK‑5), or alternatively, ALK‑1. This complex phosphorylates and activates Smad proteins that regulate transcription (3, 11, 12). Contributions of the accessory receptors Betaglycan (also known as TGF‑ beta RIII) and Endoglin, or use of Smad‑independent signaling pathways, allow for disparate actions observed in response to TGF‑ beta in different contexts (11).
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