ZBTB25 Antibody

What is ZBTB25 and why is it a significant research target?

ZBTB25 (Zinc finger and BTB domain-containing protein 25) is a transcriptional repressor protein approximately 49 kDa in size, comprising 435 amino acid residues in its canonical human form. It belongs to the broad complex, tram track, bric-a-brac/poxvirus and zinc finger (BTB/POZ) transcription family, characterized by a C-terminal DNA-binding zinc finger motif and an N-terminal protein-binding BTB/POZ domain. The zinc finger identifies and binds specific DNA sequences, while the BTB/POZ domain facilitates homodimerization/heterodimerization and interaction with other proteins . ZBTB25 is primarily localized to the nucleus and is notably expressed in hematopoietic cells and testis, with significant roles in transcriptional regulation . Its involvement in pathogen response pathways and potential as a therapeutic target for host-directed treatments (particularly for tuberculosis) makes it an important subject for immunological and infectious disease research .

What are the common synonyms and orthologs for ZBTB25?

When searching literature and databases for ZBTB25, researchers should be aware of several synonyms including KUP, ZNF46, zinc finger protein 46, zinc finger protein KUP, and C14orf51 . Understanding these alternative nomenclatures is crucial for comprehensive literature reviews and database searches. ZBTB25 gene orthologs have been identified across multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken, enabling comparative studies and the development of model systems for investigating ZBTB25 function . When designing cross-species experiments, researchers should consider potential structural and functional variations among these orthologs that might affect antibody cross-reactivity.

How does ZBTB25 function at the molecular level?

ZBTB25 functions primarily as a transcriptional repressor that associates with histone deacetylase 1 (HDAC1) and the transcriptional corepressor Sin3a to form a repressor complex . This complex binds to specific promoter regions, such as that of the IL-12B gene, inducing hypoacetylation of histone H3 and subsequently downregulating gene expression . Interestingly, ZBTB25 demonstrates dual functionality in the context of viral infections - it can promote viral RNA transcription by binding to the U-rich region of viral RNA while simultaneously suppressing cellular interferon production . The zinc finger domain of ZBTB25 is essential for its RNA-inhibitory activity through zinc ion chelation, which can be disrupted by compounds such as disulfiram, effectively repressing influenza A virus (IAV) replication .

What criteria should researchers consider when selecting a ZBTB25 antibody?

When selecting a ZBTB25 antibody, researchers should evaluate several critical factors: (1) Specificity - verify the antibody has been validated against ZBTB25 knockdown or knockout controls to confirm it recognizes the intended target; (2) Application compatibility - ensure the antibody has been validated for your specific application (Western Blot, ELISA, Immunofluorescence, etc.); (3) Species reactivity - confirm cross-reactivity with your experimental species (common reactivities include human, mouse, rat, bovine); (4) Recognition region - consider whether you need an antibody targeting specific domains (N-terminal BTB/POZ domain, middle region, or C-terminal zinc finger domain); (5) Clone type - monoclonal antibodies offer high specificity for particular epitopes, while polyclonal antibodies might provide stronger signals through multiple epitope recognition . For mechanistic studies involving protein complexes, select antibodies that won't interfere with protein-protein interactions of interest.

How can researchers validate ZBTB25 antibody specificity?

A robust validation protocol for ZBTB25 antibodies should include multiple approaches: (1) Western blot analysis comparing control versus ZBTB25 knockdown samples using siRNAs or shRNAs (such as siZBTB25-a: 5′-GGUCAUAAAUUCCCUCGAA-3′ or siZBTB25-b: 5′-GUGCAGUCCUCAAAUUUAU-3′) ; (2) Immunocytochemical staining with anti-ZBTB25 antibody compared against knockdown controls, as demonstrated in THP-1 macrophages ; (3) Reciprocal co-immunoprecipitation experiments to confirm specific protein-protein interactions, such as those between ZBTB25, HDAC1, and Sin3a ; (4) Testing antibody performance across multiple experimental conditions and sample types relevant to your research; (5) Comparison of results obtained with alternative antibodies targeting different epitopes of ZBTB25. Thorough validation ensures reliability and reproducibility in subsequent experiments.

What are the most common applications for ZBTB25 antibodies?

ZBTB25 antibodies are utilized across multiple experimental applications with varying methodological considerations: (1) Western Blot - the most common application, typically detecting the approximately 49 kDa ZBTB25 protein, with optimization of blocking conditions critical for reducing background; (2) ELISA - useful for quantitative detection in tissue or cell lysates; (3) Immunofluorescence - revealing nuclear localization of ZBTB25, often with co-staining approaches to demonstrate co-localization with other nuclear factors; (4) Chromatin Immunoprecipitation (ChIP) - for identifying ZBTB25 binding to specific promoter regions, such as the IL-12B promoter; (5) Co-immunoprecipitation - to isolate and identify ZBTB25-interacting proteins like HDAC1 and Sin3a . Researchers should optimize antibody concentrations, incubation conditions, and detection methods for each specific application to achieve optimal signal-to-noise ratios.

How should researchers design knockdown experiments to study ZBTB25 function?

When designing ZBTB25 knockdown experiments, researchers should consider: (1) Selection of knockdown technology - both shRNA and siRNA approaches have been successfully used, with documented sequences including shZBTB25-a (5′-GCTTCCATTCTGGAAAGTAAT-3′), shZBTB25-b (5′-CCAACCTGACATATTCAGCTA-3′), siZBTB25-a (5′-GGUCAUAAAUUCCCUCGAA-3′), and siZBTB25-b (5′-GUGCAGUCCUCAAAUUUAU-3′) ; (2) Validation of knockdown efficiency - using multiple methods including qRT-PCR, Western blotting, and confocal microscopy; (3) Appropriate controls - both non-targeting controls and targeting other ZBTB family members (like ZBTB2, ZBTB1, ZBTB7A) to ensure specificity; (4) Timing considerations - assess the stability of knockdown over your experimental timeframe; (5) Functional readouts - measure both direct effects (e.g., IL-12B mRNA and IL-12p40 protein levels) and downstream consequences (e.g., autophagy induction, pathogen killing) . Well-designed knockdown studies can provide robust insights into ZBTB25's role in transcriptional regulation and host-pathogen interactions.

What are the best approaches for studying ZBTB25-DNA interactions?

To investigate ZBTB25-DNA interactions, researchers should consider these methodological approaches: (1) Chromatin Immunoprecipitation (ChIP) followed by PCR or sequencing (ChIP-seq) - using antibodies against ZBTB25 to identify genome-wide binding sites or specific promoter regions, such as IL-12B ; (2) Electrophoretic Mobility Shift Assay (EMSA) - to confirm direct binding to specific DNA sequences in vitro; (3) DNA footprinting - to precisely map ZBTB25 binding sites; (4) Reporter gene assays - using constructs with wild-type and mutated putative ZBTB25 binding sites to assess functional relevance of interactions; (5) Proximity ligation assays - to visualize ZBTB25-DNA interactions in situ. When implementing these techniques, researchers should design appropriate controls and consider the potential influence of ZBTB25's interactions with other proteins (like HDAC1 and Sin3a) on its DNA-binding properties.

How can researchers effectively study ZBTB25 protein-protein interactions?

Multiple complementary approaches can be employed to characterize ZBTB25 protein-protein interactions: (1) Co-immunoprecipitation with reciprocal validation - as demonstrated for ZBTB25, HDAC1, and Sin3a interactions, using specific antibodies against each protein for precipitation followed by Western blot detection of interaction partners ; (2) Proximity-dependent labeling methods (BioID, APEX) - for identifying novel interaction partners in living cells; (3) Yeast two-hybrid screening - for systematic identification of potential interactors; (4) In vitro binding assays with recombinant proteins - to confirm direct interactions and map interaction domains; (5) Immunofluorescence microscopy with co-localization analysis - as used to visualize ZBTB25 and HDAC1 co-localization in cell nuclei . When interpreting results, researchers should consider whether interactions are direct or mediated by additional factors, and whether they are constitutive or context-dependent (e.g., infection-induced).

How is ZBTB25 implicated in M. tuberculosis infection and what methodologies reveal this role?

ZBTB25 plays a significant role in Mycobacterium tuberculosis infection through its association with the HDAC1/Sin3a repressor complex. Methodological approaches revealing this function include: (1) Co-immunoprecipitation with pHDAC1-specific antibodies followed by LC-MS/MS analysis - identifying ZBTB25 and Sin3a as HDAC1-associated proteins in infected macrophages ; (2) ChIP assays - demonstrating recruitment of ZBTB25, HDAC1, and Sin3a to the IL-12B promoter in infected cells; (3) ZBTB25 knockdown studies - showing prevention of repressor complex recruitment, enhanced IL-12B expression, and increased release of IL-12p40 from infected macrophages; (4) CFU assays - revealing reduced intracellular M. tuberculosis survival in ZBTB25-knockdown macrophages . These findings suggest ZBTB25 as a potential target for host-directed therapy against tuberculosis, particularly valuable given the emergence of drug-resistant M. tuberculosis strains.

What methodologies reveal ZBTB25's role in viral infections?

Studies investigating ZBTB25's role in viral infections, particularly influenza A virus (IAV), employ several sophisticated approaches: (1) Protein-RNA interaction studies - demonstrating ZBTB25 binding to the U-rich region of viral RNA; (2) Viral RNA polymerase activity assays - showing ZBTB25 enhancement of viral RdRp (RNA-dependent RNA polymerase) activity; (3) Functional analysis of protein domains - identifying the zinc finger domain as crucial for RNA-inhibitory activity through zinc ion chelation; (4) Pharmacological inhibition studies - revealing that disulfiram disrupts zinc finger functions and effectively represses IAV replication . Research has uncovered ZBTB25's dual transcriptional functions: promoting viral RNA transcription while suppressing cellular interferon production. These findings highlight ZBTB25 as a promising antiviral target, particularly valuable against IAV which has developed resistance to many current antiviral drugs due to its high mutation rate.

What are promising pharmacological approaches for targeting ZBTB25 in disease models?

Current research has identified several potential pharmacological approaches for targeting ZBTB25: (1) Diphenyleneiodonium (DP) - demonstrated to inhibit ZBTB25 recruitment to the IL-12B promoter at 20 μM concentration in M. tuberculosis-infected THP-1 cells, significantly reducing intracellular bacterial survival (by approximately 65%) ; (2) Disulfiram - shown to disrupt zinc finger functions of ZBTB25, effectively repressing IAV replication ; (3) Zinc chelators - potentially useful for disrupting the zinc-dependent functions of ZBTB25's zinc finger domain; (4) Targeted protein degradation approaches - emerging technologies like PROTACs could be developed to selectively degrade ZBTB25. When evaluating these approaches, researchers should assess specificity (effects on other zinc finger proteins), toxicity profiles, and efficacy across different disease models. These pharmacological strategies represent promising avenues for host-directed therapies against both bacterial (M. tuberculosis) and viral (IAV) infections.

How can researchers address background and specificity issues when using ZBTB25 antibodies?

Researchers encountering background or specificity issues with ZBTB25 antibodies should implement these troubleshooting strategies: (1) Optimization of blocking conditions - test different blocking agents (BSA, non-fat milk, commercial blockers) and concentrations to reduce non-specific binding; (2) Antibody titration - systematically test different dilutions to identify optimal signal-to-noise ratio; (3) Inclusion of validation controls - always run ZBTB25 knockdown samples alongside experimental samples to confirm signal specificity; (4) Cross-adsorption - pre-incubate antibodies with cell lysates from ZBTB25 knockout cells to remove cross-reactive antibodies; (5) Alternative fixation and permeabilization methods for immunostaining - compare different protocols to improve epitope accessibility while maintaining cellular morphology. For applications requiring highest specificity (e.g., ChIP), consider using multiple antibodies targeting different epitopes to confirm results.