WNT16 Antibody

What is WNT16 and what biological systems is it involved in?

WNT16 (Wingless-type MMTV integration site family, member 16) is a secreted glycoprotein belonging to the WNT family of signaling molecules. It plays critical roles in bone homeostasis and has been implicated in conditions such as ankylosing spondylitis (AS). Research indicates that WNT16 can regulate bone formation and influence bone loss in pathological conditions. WNT16 functions through both canonical (β-catenin-dependent) and non-canonical (JNK-dependent) signaling pathways, affecting gene expression in osteoblasts and directly inhibiting osteoclastogenesis . The protein has a calculated molecular weight of 41 kDa (365 amino acids) and is encoded by the gene with NCBI ID 51384 .

What applications are WNT16 antibodies validated for?

WNT16 antibodies have been validated for several experimental applications:

Different antibodies may have varying application validations, so researchers should refer to specific product data sheets for recommended protocols .

What species reactivity can be expected with commercial WNT16 antibodies?

The reactivity profile varies between different WNT16 antibodies:

When selecting an antibody, researchers should prioritize those with experimentally confirmed reactivity in their species of interest rather than relying solely on predicted reactivity .

What are the optimal conditions for using WNT16 antibodies in Western blotting?

For optimal Western blotting using WNT16 antibodies, researchers should:

• Use appropriate sample preparation techniques for the tissue or cell type being studied. WNT16 antibodies have successfully detected the protein in multiple cell types including HEK-293T cells, rat/mouse brain tissue, Ramos cells, Raji cells, LNCaP cells, and HepG2 cells .

• Start with recommended dilution ranges (typically 1:1000-1:8000) and optimize for your specific experimental system. As noted in product documentation: "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" .

• Use standard Western blotting protocols with appropriate blocking agents, incubation times, and detection systems. Follow specific product protocols when available.

• Include positive controls when possible. Various cell lines including HEK-293T have been validated for WNT16 expression and can serve as positive controls .

• Expect to observe bands at approximately 41 kDa, which corresponds to the observed molecular weight of WNT16 .

How can researchers effectively validate WNT16 knockdown experiments?

To validate WNT16 knockdown experiments:

• Design appropriate siRNA sequences targeting WNT16. In published research, multiple siRNA oligos have been designed to target human WNT16. For example, one study generated five siRNA oligos against human WNT16 and tested them in FOB cells .

• Transfect cells using an appropriate method (e.g., Lipofectamine 3000) and incubate for 48 hours before analyzing knockdown efficiency .

• Validate knockdown efficiency using multiple methods:

  • RT-PCR or RT-qPCR to assess mRNA levels

  • Western blotting with WNT16 antibodies to confirm protein reduction

  • Functional assays to determine if known WNT16-dependent outcomes are altered

• Include appropriate controls:

  • Non-targeting siRNA control

  • Mock transfection control

  • Untransfected control

• Consider using multiple siRNA sequences and selecting those with highest knockdown efficiency for further experiments .

What sample preparation methods are recommended for detecting WNT16 in bone tissue?

For effective detection of WNT16 in bone tissue:

• Fresh bone tissue should be carefully harvested and either snap-frozen for protein/RNA extraction or fixed appropriately for histological analysis.

• For protein extraction from bone:

  • Pulverize frozen bone tissue under liquid nitrogen conditions

  • Use strong lysis buffers containing protease inhibitors

  • Consider demineralization protocols if working with highly calcified tissues

• For RNA extraction, specialized protocols for bone tissue that address the challenges of low RNA yield and potential contamination with inhibitors should be employed.

• For histological analysis:

  • Use appropriate fixation (generally 4% paraformaldehyde)

  • Decalcify bone samples using EDTA or mild acid solutions

  • Process and embed in paraffin or prepare for frozen sectioning

  • Use antigen retrieval methods to enhance antibody binding

• For primary osteoprogenitor cell isolation (as used in WNT16 research):

  • Bone tissue from facet joints can be isolated using outgrowth methods

  • These primary cells can then be used for various assays including microarray, RT-qPCR, immunoblotting, and immunohistochemistry .

How can researchers distinguish between canonical and non-canonical WNT16 signaling pathways?

Distinguishing between canonical and non-canonical WNT16 signaling requires specific methodological approaches:

• For canonical WNT signaling assessment:

  • Measure expression levels of canonical WNT target genes such as Axin2 and Tcf1 using RT-qPCR. In WNT16-deficient mice, the expression of these genes was significantly lower in cortical bone compared to wild-type mice .

  • Use TOPflash reporter assays, which show mild but significant activation upon WNT16 treatment .

  • Analyze levels of phosphorylated LRP6 and non-phosphorylated β-catenin by Western blotting .

  • Use inhibitors like XAV939 (tankyrase inhibitor) to block β-catenin-dependent signaling by stabilizing Axin2 and the destruction complex .

• For non-canonical WNT signaling assessment:

  • Analyze phosphorylation levels of JNK and c-JUN via Western blotting. WNT16 treatment increases pJNK and p-cJUN levels in osteoblast cells .

  • Use the non-canonical WNT-specific ROR2-hTRK luciferase assay. Unlike WNT5a (a classical activator of the WNT-ROR2-JNK pathway), WNT16 did not affect this ROR2 reporter assay, suggesting it may not primarily signal through this particular non-canonical pathway .

• Compare results with positive controls:

  • WNT3a as a canonical pathway activator

  • WNT5a as a non-canonical pathway activator

What methodologies are recommended for studying WNT16's effects on osteoclastogenesis?

To study WNT16's effects on osteoclastogenesis:

• In vitro osteoclast formation assays:

  • Culture bone marrow macrophages (BMMs) with RANKL to stimulate osteoclast differentiation

  • Add recombinant WNT16 protein at various concentrations to assess inhibitory effects

  • Quantify osteoclast formation by TRAP staining (WNT16 has been shown to abolish RANKL-stimulated formation of TRAP+ multinucleated osteoclasts)

• Functional osteoclast assays:

  • Seed BMMs on bone slices and stimulate with RANKL

  • Add WNT16 at various concentrations

  • Measure resorption pit formation and CTX-I release (markers of osteoclast activity)

  • Research has demonstrated that WNT16 inhibits both TRAP+ multinucleated osteoclast formation and resorption activity

• Human osteoclast models:

  • Isolate CD14+ monocytes from human blood

  • Treat with RANKL and M-CSF to induce osteoclast differentiation

  • Add WNT16 at various doses to assess inhibitory effects

  • Studies have shown a dose-dependent inhibitory effect of WNT16 on osteoclast formation in RANKL-stimulated human CD14+ monocyte cultures

• Gene expression analysis:

  • Monitor osteoclast-specific gene expression (e.g., TRAP, cathepsin K, RANK) using RT-qPCR

  • Assess how WNT16 treatment affects the expression of these genes during osteoclast differentiation

What factors might affect WNT16 detection in experimental settings?

Several factors can influence the detection of WNT16 in research settings:

• Antibody selection and quality:

  • Different antibodies target different epitopes of WNT16 (N-terminal, C-terminal, or internal regions)

  • Polyclonal antibodies may provide broader epitope recognition but potentially lower specificity

  • Ensure antibodies are validated for your specific application and species

• Sample preparation considerations:

  • WNT16 is a secreted protein, so both cell lysates and conditioned media should be considered for analysis

  • Protein extraction methods may affect protein structure and epitope accessibility

  • Proper sample storage is essential (-20°C with glycerol for antibodies)

• Technical considerations:

  • Optimal antibody dilutions vary significantly (1:1000-1:8000 for Western blot)

  • Sample-dependent variations necessitate optimization for each experimental system

  • Antigen retrieval methods for IHC may need optimization for bone tissues

• Biological variables:

  • Expression levels of WNT16 vary across different tissues and cell types

  • Disease states (like ankylosing spondylitis) may alter WNT16 expression

  • Cell culture conditions can affect WNT16 expression levels

How should researchers interpret changes in WNT16 expression in bone disorders?

When interpreting WNT16 expression changes in bone disorders:

• Consider the specific bone pathology being studied:

  • In ankylosing spondylitis (AS), WNT16 expression is significantly elevated in AS osteoprogenitor cells compared to controls

  • WNT16-deficient mice develop spontaneous fractures due to low cortical thickness and high cortical porosity, indicating its importance in normal bone homeostasis

• Analyze both protein and mRNA levels:

  • Validate findings using multiple detection methods

  • Correlate expression with functional outcomes (e.g., bone formation activity, cell senescence)

• Consider cellular context:

  • WNT16 treatment inhibits bone formation in AS-osteoprogenitor cells but not in control cells

  • WNT16 treatment increases cell senescence markers (β-galactosidase staining) in AS-osteoprogenitor cells

  • Under H₂O₂ stress-induced premature senescence conditions, WNT16 treatment increases cell senescence, p21 protein, and senescence-associated secretory phenotype (SASP) mRNA expression in AS-osteoprogenitor cells

• Evaluate pathway activation:

  • Assess both canonical (β-catenin-dependent) and non-canonical (JNK-dependent) WNT signaling activation

  • Determine if observed effects are direct or mediated through other factors (e.g., OPG)

• Consider therapeutic implications:

  • In temporomandibular joint osteoarthritis, intra-articular injection of Wnt pathway inhibitor (SM04690) upregulates WNT16 expression and reduces disease progression

What controls should be included when using WNT16 antibodies in research?

Essential controls for WNT16 antibody experiments include:

• Positive controls:

  • Cell lines with confirmed WNT16 expression (HEK-293T, Ramos, Raji, LNCaP, HepG2 cells)

  • Tissue samples with known WNT16 expression (mouse/rat brain tissue)

• Negative controls:

  • WNT16 knockout or knockdown samples when available

  • Secondary antibody-only controls to assess non-specific binding

  • Isotype controls (Rabbit IgG for many WNT16 antibodies)

• Specificity controls:

  • Blocking peptide competition assays with the immunizing peptide

  • Multiple antibodies targeting different epitopes of WNT16 to confirm specificity

• Procedural controls:

  • Loading controls for Western blot (β-actin, GAPDH)

  • Housekeeping genes for RT-qPCR normalization

  • Tissue-specific markers for IHC to confirm tissue integrity

• Biological reference controls:

  • Compare diseased vs. healthy tissues (e.g., AS patient samples vs. disease controls)

  • Age-matched and sex-matched controls for in vivo studies

How does WNT16 influence cell senescence in pathological conditions?

Recent research has revealed important connections between WNT16 and cell senescence:

• In ankylosing spondylitis (AS):

  • WNT16 expression is significantly elevated in AS osteoprogenitor cells compared to controls

  • WNT16 treatment increases cell senescence markers in AS-osteoprogenitor cells, as evidenced by β-galactosidase staining

  • Under H₂O₂ stress-induced premature senescence conditions, WNT16 treatment increases:

    • Cell senescence in AS-osteoprogenitor cells

    • p21 protein expression (cell cycle regulator associated with senescence)

    • Senescence-associated secretory phenotype (SASP) mRNA expression

• Mechanistic insights:

  • WNT16-induced SASP expression in AS-osteoprogenitor cells was reduced in WNT16 knockdown cultures, confirming the specificity of this effect

  • This suggests that elevated WNT16 may contribute to the pathophysiology of AS through induction of cell senescence

• Functional consequences:

  • WNT16 treatment inhibits bone formation in AS-osteoprogenitor cells but not in control cells

  • This selective effect suggests that WNT16 signaling outcomes may differ depending on the cellular context and disease state

What are the dual roles of WNT16 in osteoblast and osteoclast regulation?

WNT16 exhibits complex regulatory functions in bone homeostasis:

• Effects on osteoblasts (bone-forming cells):

  • WNT16 activates canonical WNT signaling in osteoblasts, as evidenced by:

    • Increased expression of canonical target genes (Axin2 and Tcf1)

    • Activation of TOPflash reporter

    • Increased levels of phosphorylated LRP6 and non-phosphorylated β-catenin

  • WNT16 also activates non-canonical JNK signaling in osteoblasts

    • Increased levels of phosphorylated JNK and c-JUN

  • Context-dependent effects:

    • In normal osteoblasts, WNT16 may promote bone formation

    • In AS-osteoprogenitor cells, WNT16 inhibits bone formation and promotes cell senescence

• Direct effects on osteoclasts (bone-resorbing cells):

  • WNT16 directly inhibits osteoclastogenesis:

    • Abolishes RANKL-stimulated formation of TRAP+ multinucleated osteoclasts in mouse BMM cultures

    • Inhibits RANKL-stimulated CTX-I release and resorption pit formation

    • Shows dose-dependent inhibition of osteoclast formation in human CD14+ monocyte cultures

  • These effects occur in the absence of osteoblasts, indicating direct action on osteoclast precursors

• Indirect effects on osteoclasts:

  • WNT16 may also regulate osteoclast formation indirectly through effects on osteoblasts

  • The dual regulation of bone formation and resorption positions WNT16 as a key regulator of bone homeostasis