ZNRF2 Antibody

What is ZNRF2 and why is it significant for biological research?

ZNRF2 (Zinc And Ring Finger 2) is an E3 ubiquitin ligase that plays critical roles in multiple cellular processes. Its significance stems from:

• Function as an E3 ubiquitin ligase that accepts ubiquitin from E2 ubiquitin-conjugating enzymes and transfers it to targeted substrates

• Regulation of the Na+/K+ ATPase (sodium-potassium pump), which is essential for maintaining cellular electrochemical gradients

• Involvement in growth factor and insulin signaling pathways through phosphorylation-dependent membrane localization

• Potential oncogenic roles in multiple human cancers, including non-small cell lung cancer and hepatocellular carcinoma

• Interaction with mTOR (mammalian target of rapamycin) signaling pathway, suggesting involvement in nutrient sensing and cellular growth regulation

For effective investigation of these biological functions, high-quality ZNRF2 antibodies are essential research tools.

What are the typical applications for ZNRF2 antibodies in research?

ZNRF2 antibodies can be employed in multiple experimental techniques:

Note: Dilution recommendations may vary between antibody sources and should be optimized for specific experimental conditions .

What is the expected molecular weight of ZNRF2 in Western blot analysis?

When using ZNRF2 antibodies for Western blotting, researchers should note:

• Calculated molecular weight: approximately 24 kDa

• Observed molecular weight: typically 30-35 kDa

The discrepancy between calculated and observed molecular weights may result from:

• Post-translational modifications, particularly N-myristoylation

• Phosphorylation at multiple sites (including Ser19, Ser82, and Ser145)

• Sample preparation conditions and gel system differences

How should sample preparation be optimized for ZNRF2 detection?

Effective sample preparation for ZNRF2 detection requires consideration of its subcellular localization:

• Membrane-associated versus cytosolic ZNRF2:

  • N-myristoylated ZNRF2 localizes to intracellular membranes and plasma membrane

  • Phosphorylation by growth factors (IGF1, serum) or other stimuli (PMA, forskolin) releases ZNRF2 into the cytosol

  • For comprehensive analysis, both membrane and cytosolic fractions should be prepared by ultracentrifugation of detergent-free lysates

• Lysis buffer recommendations:

  • For total ZNRF2: Use buffers containing detergents capable of solubilizing membrane proteins

  • For fractionation studies: Use detergent-free buffers followed by ultracentrifugation to separate membrane and soluble fractions

  • Addition of phosphatase inhibitors is critical when studying phosphorylated forms of ZNRF2

• Effect of stimuli on ZNRF2 localization:

  • Serum starvation increases membrane-associated ZNRF2

  • Growth factor stimulation (IGF1, serum) decreases membrane-associated ZNRF2

  • PI3K inhibitors (PI-103, GDC-0941) and PKB inhibitors (MK-2206, AKTi-1/2) reverse growth factor effects

What controls should be included when using ZNRF2 antibodies?

For rigorous validation of ZNRF2 antibody specificity:

• Positive controls: Rat brain tissue, mouse brain tissue, mouse testis tissue, and mouse kidney tissue express detectable levels of endogenous ZNRF2

• Negative controls: ZNRF2 knockdown/knockout samples provide the most stringent negative control

• Mutation controls: For localization studies, the non-myristoylated G2A mutant of ZNRF2 can serve as a control for membrane localization

• Peptide competition: Pre-incubation of antibody with immunizing peptide should abolish specific signal

How can phospho-specific ZNRF2 antibodies be utilized to study signaling pathways?

Phospho-specific antibodies recognizing phosphorylated ZNRF2 sites provide valuable tools for studying its regulation:

• Key phosphorylation sites:

  • Ser19: Phosphorylated by PKBα, SGK1, p90RSK, and PKA

  • Ser82: Phosphorylated by p90RSK and PKA

  • Ser145: Phosphorylated by PKA

• Experimental approach:

  • Use phospho-specific antibodies to monitor site-specific phosphorylation after stimulation with growth factors (IGF1), serum, PMA, or forskolin

  • Apply kinase inhibitors (PI-103, Gö6983, H-89) to block specific signaling pathways

  • Combine with subcellular fractionation to correlate phosphorylation status with membrane/cytosol distribution

• Data interpretation:

  • Phosphorylation at Ser19 and Ser145 correlates with cytosolic localization

  • Inhibition of relevant kinases increases membrane association

  • Temporal analysis can reveal kinetics of phosphorylation and subcellular translocation

What methodologies can elucidate ZNRF2's role in cancer progression?

Given ZNRF2's potential oncogenic roles, several approaches using ZNRF2 antibodies can provide insights:

• Tissue expression analysis:

  • IHC with ZNRF2 antibodies on tumor tissue microarrays to assess expression in different cancer types

  • Correlation with clinical parameters (stage, grade, patient outcome)

• Functional studies:

  • Combine ZNRF2 knockdown (validated by Western blot) with proliferation assays

  • In HCC research, ZNRF2 siRNA limited HepG2 cell proliferation

  • Monitor effects on downstream pathways using phospho-specific antibodies for targets in mTOR signaling

• Interaction studies:

  • Immunoprecipitation with ZNRF2 antibodies to identify cancer-relevant binding partners

  • Analysis of interaction with mTOR complex components in different cancer models

How can researchers investigate ZNRF2-mediated ubiquitination processes?

To study ZNRF2's E3 ubiquitin ligase activity:

• In vitro ubiquitination assays:

  • Immunoprecipitate ZNRF2 using specific antibodies

  • Combine with purified E1, E2 (predominantly Ubc13/Uev1a for Lys63-linkages), ubiquitin, and potential substrates

  • Detect ubiquitinated products by Western blotting

• Substrate identification:

  • Known substrate: Na+/K+ATPase α1 subunit (via UBZ domain interaction)

  • For novel substrates: Combine ZNRF2 immunoprecipitation with mass spectrometry

  • Validate by detecting increased ubiquitination of potential substrates when ZNRF2 is overexpressed

• Domain-specific functions:

  • RING domain: Interacts with E2 enzymes (mainly Ubc13)

  • UBZ domain: Mediates substrate recognition (e.g., Na+/K+ATPase α1)

Why might ZNRF2 antibodies show variable signal intensity in Western blots?

Several factors can affect ZNRF2 detection:

• Protein localization variability:

  • Growth factor stimulation causes ZNRF2 translocation from membranes to cytosol

  • Serum starvation increases membrane-associated ZNRF2

  • Ensure consistent culture conditions and stimulation protocols

• Post-translational modifications:

  • N-myristoylation affects membrane association

  • Phosphorylation status changes with cellular signaling

  • Use phosphatase inhibitors in lysis buffers when studying total ZNRF2

• Technical considerations:

  • Observed molecular weight (30-35 kDa) differs from calculated (24 kDa)

  • Transfer efficiency for membrane proteins may require optimization

  • Blocking conditions might need adjustment for different antibodies

What are the best methods to validate ZNRF2 antibody specificity?

For rigorous validation:

• Genetic approaches:

  • ZNRF2 knockdown (siRNA/shRNA) should reduce signal intensity

  • ZNRF2 knockout samples provide definitive negative controls

  • Reintroduction of ZNRF2 should restore signal

• Multiple antibody validation:

  • Compare results using antibodies targeting different epitopes of ZNRF2

  • Antibodies raised against different regions (e.g., N-terminal vs. C-terminal) should yield consistent patterns

• Cross-reactivity assessment:

  • Test for potential cross-reactivity with the paralog ZNRF1

  • Comparison of expression patterns with mRNA data from public databases

How can ZNRF2 antibodies help investigate its interaction with mTOR signaling?

Recent findings highlight ZNRF2's interaction with mTOR, suggesting novel research directions:

• Protein complex identification:

  • Immunoprecipitation with ZNRF2 antibodies followed by mass spectrometry identified mTOR as a binding partner

  • N-myristoylation of ZNRF2 is critical for mTOR interaction

• Experimental approaches:

  • Co-immunoprecipitation using ZNRF2 antibodies to pull down mTOR complex components

  • Immunofluorescence to study co-localization of ZNRF2 with mTOR

  • Phospho-specific antibodies to monitor mTOR activity in response to ZNRF2 manipulation

• Biological questions to address:

  • Does ZNRF2 regulate mTOR activity through ubiquitination?

  • How does ZNRF2 phosphorylation affect mTOR interaction?

  • Are genetic mutations in ZNRF2 involved in mTOR-driven cancers?

What methodological approaches can reveal ZNRF2's role in neuronal function?

Given ZNRF2's abundance in brain tissue and potential neuronal functions:

• Expression analysis:

  • Immunohistochemistry and immunofluorescence in brain sections

  • Co-localization with neuronal markers

• Functional studies:

  • Investigation of ZNRF2's role in "establishment and maintenance of neuronal transmission and plasticity"

  • Analysis of effects on neuronal Na+/K+ATPase function

  • Study of ZNRF2's impact on neuronal response to ischemia/reperfusion

• Technical considerations:

  • Rat and mouse brain tissues provide reliable positive controls

  • Combine with neuronal-specific markers for co-localization studies

  • Consider cell type-specific expression patterns within neural tissues