Detection of Human and Mouse Wnt‑3a by Western Blot. Western blot shows lysates of BG01V human embryonic stem cells and mouse lung tissue. PVDF membrane was probed with 2 µg/mL of Rat Anti-Human/Mouse Wnt‑3a Monoclonal Antibody (Catalog # MAB1324) followed by HRP-conjugated Anti-Rat IgG Secondary Antibody (Catalog # HAF005). A specific band was detected for|
Wnt‑3a at approximately 40 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
Intracellular Staining by Flow Cytometry
|Detection of Wnt-3a in BG01V Human Stem Cells by Flow Cytometry. BG01V human embryonic stem cells were stained with Rat Anti-Human/Mouse Wnt-3a Monoclonal Antibody (Catalog # MAB1324, filled histogram) or isotype control antibody (Catalog # MAB006, open histogram), followed by Allophycocyanin-conjugated Anti-Rat IgG Secondary Antibody (Catalog # F0113). To facilitate intracellular staining, cells were fixed and permeabilized with FlowX FoxP3 Fixation & Permeabilization Buffer Kit (Catalog # FC012).|
Alkaline Phosphatase Production Induced by|
Wnt‑3a and Neutralization by Human/Mouse Wnt‑3a Antibody. Recombinant Mouse Wnt‑3a (Catalog # 1324-WN) induces alkaline phosphatase production in the MC3T3‑E1 mouse preosteoblast cell line in a dose-dependent manner (orange line). Under these conditions, alkaline phosphatase production elicited by Recombinant Mouse Wnt‑3a (10 ng/mL) is neutralized (green line) by increasing concentrations of Rat Anti-Human/Mouse Wnt‑3a Monoclonal Antibody (Catalog # MAB1324). The ND50 is typically 3-15 µg/mL.
Wnt-3a is one of about 19 vertebrate members of the Wingless-type MMTV integration site (Wnt) family of highly conserved cysteine-rich secreted glycoproteins important for normal developmental processes (1‑3). Wnts bind to receptors of the Frizzled family in conjunction with a coreceptor of the low-density lipoprotein receptor-related protein family (LRP-5 or -6), or the Ryk atypical receptor tyrosine kinase (1, 4). Mouse Wnt-3a is a 44 kDa secreted hydrophobic glycoprotein containing a conserved pattern of 24 cysteine residues (5). Like other Wnts, Wnt-3a is modified by palmitate addition (at Cys 77) following glycosylation, which increases its hydrophobicity, secretion and activity (6, 7). A second site at Ser 209 is modified by palmitoleic acid and also contributes to activity and secretion (8). Mouse Wnt-3a shares 96% amino acid (aa) identity with human Wnt-3a, and 97% with bovine and canine Wnt-3a. The rat Wnt-3a precursor as it is apparently translated shares 100% aa identity with mouse Wnt-3a aa 63-352 (9). Wnt-3a also shares 87% aa identity with Wnt-3. During development, Wnt-3a is morphogen that is thought to coordinate somitogenesis and mesoderm boundary determination, and is expressed at the same locations and times as Wnt-2b and Wnt-5a (10). When Wnt-3a is deleted, mice fail to develop a hippocampus, and show defects in anterior-posterior patterning, somite development and tailbud formation (10-13). Recombinant Wnt-3a promotes proliferation of committed stem cell lineages in vitro, and may help maintain the cells in an undifferentiated state (6, 14) For example, Wnt-3a can induce self-renewal of hematopoietic stem cells, allowing expansion without further differentiation (6).
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