|Detection of Mouse Nidogen‑2 by Western Blot. Western blot shows lysates of mouse placenta tissue. PVDF membrane was probed with 2 µg/mL of Rat Anti-Mouse Nidogen‑2 Monoclonal Antibody (Catalog # MAB6760) followed by HRP-conjugated Anti-Rat IgG Secondary Antibody (Catalog # HAF005). Specific bands were detected for Nidogen‑2 at approximately 120-200 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.|
Nidogen-2 (also named Entactin-2) is a 160-200 kDa, secreted, monomeric basement membrane glycoprotein (1,2). Nidogens 1 and 2 are expressed in nearly all basement membranes (1-4) where they interact with Laminins, Collagen type IV and proteoglycan family members to form structural scaffolds (5, 6). Mice deleted for both Nidogen genes die at birth due to delayed basement membrane maturation of the lungs and heart (1). However, deletion of one Nidogen gives a mild phenotype, indicating that their functions overlap (1, 7). In adult, muscle Nidogen-2 expression is concentrated in the basal lamina of synapses and is required for their maturation and maintenance (8). Both Nidogens bind Laminin, Perlecan and Collagens I and IV, but only Nidogen-1 binds Fibulins (1, 4). The two Nidogens share approximately 40% amino acid (aa) identity in mouse and are structurally similar (1, 2, 5). Cleavage of a 30 aa signal sequence from mouse Nidogen-2 produces a 1373 aa mature protein containing three globular domains (G1-3) separated by a link region and an extended rod-shaped segment. The G1 domain is reported to bind type IV Collagen, the G2 Nidogen ( beta -barrel) domain interacts with Perlecan, and the C-terminal G3 beta -propeller structure is associated with Laminin binding. The mucin-like link region is longer in Nidogen-2 than Nidogen-1, and contains both N- and O-glycosylation (3, 8). It contains one EGF-like motif, and an RGD motif that binds alpha 3 beta 1 Integrins. The rod-shaped segment contains four additional EGF-like motifs, two of which bind calcium, and two thyroglobulin type 1 domains that serve as a binding site for alpha v beta 3 Integrins. If species‑specific insertions and deletions are disregarded, mature mouse Nidogen-2 shares 80%, 92%, 74%, 73%, 71% and 68% aa identity with human, rat, bovine, porcine, canine and equine Nidogen-2, respectively.