Zc3h12a Antibody
Description
Biological Function of ZC3H12A
ZC3H12A regulates mRNA stability and immune homeostasis by degrading transcripts encoding pro-inflammatory cytokines (e.g., IL6, IL12B) via their 3'-UTRs . It also modulates apoptosis and angiogenesis in cancer . Deficiency in ZC3H12A leads to severe autoimmune disorders, anemia, and dysregulated cytokine production in mice .
Cancer Biomarker Potential
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Colorectal Cancer (CRC):
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ZC3H12A mRNA and protein levels are significantly elevated in stage I CRC compared to advanced stages (P < 0.001) .
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Low ZC3H12A correlates with aggressive tumor features (lymph node metastasis, P = 0.0008) and shorter disease-free survival (HR = 0.41, P = 0.0128) .
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IHC validation in 110 CRC samples confirmed higher ZC3H12A expression in stage I tumors vs. normal tissues (P = 0.0156) .
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Immune Regulation
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ZC3H12A-deficient macrophages overproduce IL-6 and IL-12p40 due to impaired mRNA decay .
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In Zc3h12a<sup>−/−</sup> mice, IL-17-induced pulmonary inflammation is exacerbated, with elevated neutrophil infiltration and CXCL5 levels .
Therapeutic Implications
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ZC3H12A suppresses tumor metastasis by inhibiting angiogenesis and EMT pathways .
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Its RNase activity destabilizes transcripts of immune checkpoint molecules (e.g., PD-L1) .
Technical Considerations for Antibody Use
Key Research Insights
Product Specs
Target Background
The Zc3h12a antibody targets Regnase-1, an endoribonuclease with diverse roles in various biological processes, including:
- Inflammation and Immune Homeostasis: Regnase-1 modulates the inflammatory response by degrading cytokine-induced inflammation-related mRNAs (e.g., IL6 and IL12B) during early inflammation. It prevents aberrant T-cell mediated immune reactions by degrading mRNAs associated with T-cell activation (e.g., IL6, IL2, ICOS, TNFRSF4, TNFR2, and REL). It cooperates with ZC3H12A to inhibit Th17 cell differentiation, repressing mRNAs encoding Th17 cell-promoting factors (IL6, ICOS, REL, IRF4, NFKBID, and NFKBIZ). It also participates in a TANK-dependent negative feedback response to attenuate NF-κB activation. Regnase-1 influences macrophage polarization, promoting the shift from pro-inflammatory M1 to anti-inflammatory M2 macrophages. Furthermore, it plays a key role in negatively regulating macrophage-mediated inflammatory response and immune homeostasis by positively regulating deubiquitinase activity.
- Angiogenesis: Regnase-1 promotes angiogenesis by inhibiting the production of antiangiogenic microRNAs.
- Adipogenesis: Regnase-1 is involved in the process of adipogenesis.
- Apoptosis: Regnase-1 influences cardiomyocyte cell death and promotes macrophage apoptosis under stress conditions.
- mRNA Decay: Regnase-1 functions as an endoribonuclease involved in mRNA decay, targeting mRNAs with a stem-loop structure, often in their 3'-UTRs. This activity is partly dependent on the helicase UPF1.
- MicroRNA Biogenesis: Regnase-1 inhibits microRNA biogenesis by cleaving precursor miRNAs.
- Protein Ubiquitination: Regnase-1 affects the overall ubiquitination of cellular proteins, positively regulating deubiquitinase activity and preventing JNK and NF-κB signaling pathway activation.
- Glial Differentiation: Regnase-1 positively regulates glial differentiation of neuroprogenitor cells via an APP-dependent pathway.
- Septic Myocardial Contractile Dysfunction: Regnase-1 attenuates septic myocardial contractile dysfunction by reducing IKK-mediated NF-κB activation.
- Stress Granule Formation: Regnase-1 prevents stress granule formation.
Regnase-1 also exhibits self-regulation by destabilizing its own mRNA.
Further research has elucidated several key aspects of Regnase-1 function, as detailed in the following publications:
- Regnase-1 predominantly regulates mTORC1 signaling. PMID: 30297433
- MCPIP1 (Regnase-1) acts as a potent host defense against Coxsackievirus B3 infection and viral myocarditis. PMID: 29043433
- MCPIP1 overexpression modulates miRNA levels in adipocytes, impacting differentiation. PMID: 28939056
- Duodenal Regnase-1 controls PHD3 expression, affecting duodenal iron uptake. PMID: 28538180
- Regnase-1 and Roquin function non-redundantly in T-cell activation; Regnase-1 represses Roquin mRNA. PMID: 29127149
- MCPIP1 plays a significant role in early cortical neurogenesis. PMID: 27523618
- MCPIP1 negatively regulates psoriatic skin inflammation through IL-17A and IL-17C. PMID: 27920272
- IL-17A-mediated induction of MCPIP1 regulates gene expression in psoriatic epidermis. PMID: 27180111
- Regnase-1 RNase activity is tightly controlled by intra- and intermolecular interactions. PMID: 26927947
- MCPIP1 overexpression induces apoptosis. PMID: 26833120
- MCPIP1 is a target of mmu-miR-27-5p. PMID: 26295043
- MCPIP1-MSCs express increased levels of proteins involved in angiogenesis, autophagy, and differentiation. PMID: 26214508
- MCPIP1 mediates minocycline-induced protection from brain ischemia. PMID: 25888869
- MCPIP1 knockdown enhances IL-17-mediated signaling. PMID: 26320658
- MCPIP1 protects against adverse cardiac remodeling after myocardial infarction. PMID: 25840774
- STAT6 and KLF4 mediate IL-4-induced M2 polarization via MCPIP1's catalytic activities. PMID: 25934862
- Regnase-1 and Roquin differentially regulate mRNAs based on translation status. PMID: 26000482
- Regnase-1 is a key regulator of immune responses. PMID: 24163394
- Roquin and Regnase-1 repress Th17 cell-promoting factors. PMID: 25282160
- MCPIP1 deficiency causes severe anemia related to autoimmune mechanisms. PMID: 24324805
- Hematopoietic MCPIP1 deficiency leads to systemic inflammation but diminished atherogenesis. PMID: 24223214
- MCPIP1 is a key regulator of adipogenesis. PMID: 24418043
- MCPIP1 is a potent regulator of inflammation and immune homeostasis. PMID: 23567898
- MCPIP1 deficiency affects electroacupuncture-induced cerebral protection. PMID: 23663236
- MCPIP1 selectively suppresses TLR4 signaling and protects against septic shock. PMID: 23422584
- MCPIP1 is regulated by IL-17 and IL-1. PMID: 23658019
- Dynamic control of Regnase-1 expression in T cells is critical for controlling T-cell activation. PMID: 23706741
- MCPIP1 is implicated in rheumatoid arthritis and endothelial dysfunction. PMID: 23580143
- MCPIP1 is a potent regulator of innate immunity involved in infective diseases. PMID: 22777400
- MCPIP1 induces adipogenesis via reactive oxygen/nitrogen species and ER stress. PMID: 22739135
- MCPIP1 absence exacerbates ischemic brain damage. PMID: 22196138
- MCPIP1 coordinates stress granule formation and apoptosis. PMID: 21971051
- MCPIP1 has therapeutic potential for protecting the heart from inflammatory pathologies. PMID: 21616078
- MCPIP1 negatively regulates JNK and NF-κB activity by deubiquitination. PMID: 21115689
- MCPIP1 (Zc3h12a) regulates macrophage activation. PMID: 18178554
- Zc3h12a prevents immune disorders by controlling inflammatory gene stability. PMID: 19322177
- MCP-1-induced protein (MCPIP1) induces adipogenesis independently of PPARγ. PMID: 19666473
- MCPIP1 is a novel RNase regulating inflammatory responses via IL-6 mRNA. PMID: 19894584
Q&A
How should researchers validate ZC3H12A antibodies across multiple experimental applications?
Validation requires parallel testing in the intended application (e.g., Western blot [WB], immunohistochemistry [IHC], flow cytometry) using positive and negative controls. For WB, lysates from cell lines with confirmed ZC3H12A expression (e.g., K-562, Raji, or HeLa cells) should show a single band at 66 kDa, aligning with its calculated molecular weight . Immunofluorescence (IF) validation in U-251 cells should reveal cytoplasmic and nuclear localization, consistent with ZC3H12A’s dual subcellular distribution . For IHC, antigen retrieval with TE buffer (pH 9.0) or citrate buffer (pH 6.0) is critical to unmask epitopes in formalin-fixed tissues . Researchers must compare results across at least two independent antibody clones (e.g., monoclonal vs. polyclonal) to confirm specificity.
How does species reactivity impact experimental design?
ZC3H12A antibodies with cross-species reactivity (e.g., human, mouse, rat) enable comparative studies in translational models. For example, Proteintech 25009-1-AP detects ZC3H12A in mouse spleen and rat kidney tissues, facilitating knockout (KO) validation in murine models . In contrast, antibodies with restricted reactivity (e.g., human-only) require careful matching of experimental models to avoid false negatives. Researchers should verify reactivity using species-specific positive controls, such as lysates from ZC3H12A-transfected cell lines.
How can discrepancies between observed and expected molecular weights be resolved?
While ZC3H12A’s calculated molecular weight is 66 kDa, post-translational modifications (e.g., phosphorylation, ubiquitination) or alternative splicing may alter migration patterns. If multiple bands appear in WB, researchers should:
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Perform peptide competition assays using the immunogen (e.g., ZC3H12A fusion protein Ag13877) .
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Use KO controls (e.g., Zc3h12a−/− cells) to identify nonspecific bands .
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Optimize gel electrophoresis conditions (e.g., 10–12% SDS-PAGE) to improve resolution .
What methodological strategies are employed to study ZC3H12A’s RNase activity in vitro?
ZC3H12A degrades mRNA substrates via its PIN-like RNase domain. To assay this activity:
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RNA immunoprecipitation (RIP): Use IP-validated antibodies (e.g., Proteintech 25009-1-AP) to pull down ZC3H12A-RNA complexes, followed by qPCR or sequencing to identify target transcripts .
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In vitro RNase assays: Recombinant ZC3H12A is incubated with radiolabeled RNA probes, and degradation is measured via gel electrophoresis. Antibodies like Bio-Techne NBP3-18333 (validated in ELISA) can quantify protein levels in parallel .
How do researchers address cross-reactivity with ZC3H12 family members?
ZC3H12A shares structural homology with ZC3H12B-D, necessitating stringent validation. Approaches include:
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Knockdown/rescue experiments: siRNA-mediated silencing of ZC3H12A followed by overexpression of wild-type or mutant protein.
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Selective epitope mapping: Monoclonal antibodies targeting non-conserved regions (e.g., N-terminal domains) minimize off-target binding .
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Multiplexed Western blotting: Simultaneous probing with ZC3H12A-specific and pan-ZC3H12 antibodies to distinguish signals .
What steps mitigate nonspecific staining in IHC?
Nonspecific staining often arises from improper antigen retrieval or antibody concentration. Key adjustments:
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Antigen retrieval: Compare TE buffer (pH 9.0) and citrate buffer (pH 6.0) to optimize epitope exposure .
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Titration: Test antibody dilutions between 1:250 and 1:1000, using negative control tissues (e.g., Zc3h12a−/− mice) to establish baseline noise .
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Blocking: Use 5% non-fat dry milk or serum matching the antibody host species to reduce background .
How is ZC3H12A knockout validation performed in murine models?
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Genotyping: Confirm Zc3h12a disruption via PCR using primers flanking the targeted exon.
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Phenotypic analysis: Assess immune dysregulation (e.g., lymphadenopathy, elevated TNF-α) as seen in dendritic cell-specific KO models .
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Antibody validation: Ensure the antibody does not cross-react with residual truncated proteins by WB and IHC .
Comparative Antibody Performance Table
| Antibody Clone | Host/Isotype | Applications Validated | Reactivity | Key Strengths |
|---|---|---|---|---|
| Proteintech 84521-5-RR | Rabbit/IgG | WB, IF/ICC, FC (Intra) | Human | Recombinant format minimizes lot variability |
| Proteintech 25009-1-AP | Rabbit/IgG | WB, IHC, IP, IF | Human, Mouse, Rat | Broad species reactivity; KO-validated |
| Bio-Techne NBP3-18333 | Rabbit/IgG | WB, IF, IHC | Human, Mouse | BSA-free formulation reduces background |
| Proteintech 68616-1-PBS | Mouse/IgG1 | WB, Indirect ELISA | Human | Monoclonal specificity for epitope mapping |
How does ZC3H12A regulate immune responses in dendritic cells?
ZC3H12A degrades pro-inflammatory cytokine mRNAs (e.g., IL-6, TNF-α), preventing excessive immune activation. In dendritic cell-specific KO models, loss of ZC3H12A leads to TNF-α-driven lymphadenopathy, mimicking autoimmune phenotypes . Researchers can model this by transfecting DCs with siRNA and measuring cytokine secretion via ELISA.
What experimental models elucidate ZC3H12A’s role in ischemic heart disease?
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Hypoxia-reoxygenation assays: Cardiomyocytes exposed to low oxygen followed by reoxygenation show upregulated ZC3H12A, detectable via WB using antibodies validated in ischemic tissues .
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Transcriptomic profiling: RIP-seq with ZC3H12A antibodies identifies cardiac-enriched mRNA targets, linking its RNase activity to ischemic injury pathways .
