ZHX2 Antibody

What is ZHX2 and why is it an important research target?

ZHX2 is a transcription factor containing zinc finger motifs and five homeodomains involved in DNA-protein and protein-protein interactions. It functions primarily as a transcriptional repressor by reducing expression of genes including cyclin A, cyclin E, and alpha-fetoprotein . ZHX2 exhibits context-dependent roles - functioning as a tumor suppressor in hepatocellular carcinoma, lymphoma, and myeloma, while acting as an oncogene in clear cell renal carcinoma (ccRCC) and triple-negative breast cancer (TNBC) . Additionally, ZHX2 has emerged as a critical regulator in metabolic conditions, especially diabetes-induced liver injury, and in immune cell function regulation .

The choice between monoclonal and polyclonal ZHX2 antibodies depends on your experimental goals:

Monoclonal antibodies (e.g., 68268-1-Ig) offer:

• Higher specificity for a single epitope

• Greater lot-to-lot consistency

• Preferred for quantitative applications where reproducibility is critical

• Optimal for distinguishing between closely related proteins

Polyclonal antibodies (e.g., 20136-1-AP) provide:

• Recognition of multiple epitopes on ZHX2

• Higher sensitivity for detection of low-abundance targets

• Greater resistance to antigen changes from denaturation or fixation

• Better for applications like IP where binding to multiple epitopes is advantageous

For studies examining post-translational modifications or specific ZHX2 isoforms, choose antibodies with epitopes in regions that will not be affected by these variations .

What are the optimal conditions for western blot detection of ZHX2?

For effective western blot detection of ZHX2:

• Sample preparation: Use RIPA buffer with protease inhibitors for tissue/cell lysis.

• Loading amount: Load 20-40 μg of total protein per lane.

• Gel selection: Use 8% SDS-PAGE gels due to ZHX2's molecular weight (92-100 kDa) .

• Transfer conditions: Transfer to PVDF membrane at 100V for 90 minutes (wet transfer).

• Blocking solution: 5% non-fat milk in TBST for 1 hour at room temperature.

• Primary antibody dilution:

  • For polyclonal antibodies: 1:1000-1:5000 in 5% BSA

  • For monoclonal antibodies: 1:5000-1:50000 in 5% BSA

• Incubation conditions: Overnight at 4°C with gentle rocking.

• Detection method: HRP-conjugated secondary antibodies with ECL substrate.

The observed molecular weight of ZHX2 typically ranges from 92-100 kDa, though this may vary slightly between different cell types and tissues .

What protocol modifications are needed for successful ZHX2 immunohistochemistry?

For optimal ZHX2 detection in tissue sections:

• Fixation: 10% neutral-buffered formalin (24 hours) for paraffin-embedded sections.

• Antigen retrieval: This is critical for ZHX2 detection.

  • Primary option: TE buffer pH 9.0, pressure cooking for 3 minutes

  • Alternative: Citrate buffer pH 6.0, heat-induced for 15-20 minutes

• Blocking: 10% normal serum (matched to secondary antibody species) with 1% BSA for 1 hour.

• Primary antibody:

  • For paraffin sections: 1:50-1:500 for polyclonal (20136-1-AP)

  • For paraffin sections: 1:250-1:1000 for monoclonal (68268-1-Ig)

• Incubation time: Overnight at 4°C in a humidified chamber.

• Detection system: DAB or AEC-based detection systems work well.

• Controls: Include tissue known to express ZHX2 (brain tissue is recommended as a positive control) .

• Counterstaining: Hematoxylin counterstaining should be light to avoid masking nuclear ZHX2 signal.

For frozen sections, fixation in cold acetone for 10 minutes prior to staining is recommended .

How can researchers validate ZHX2 antibody specificity?

Validation of ZHX2 antibody specificity is essential to ensure reliable results:

• Positive control samples: Use tissues or cells with confirmed ZHX2 expression (SH-SY5Y cells, HEK-293 cells, LNCaP cells, brain tissues) .

• Peptide competition assay: Pre-incubate antibody with immunizing peptide before application to verify signal reduction.

• Knockdown/Knockout validation: Use siRNA/shRNA knockdown or CRISPR knockout cells as negative controls. Several published studies have used ZHX2 shRNAs (e.g., sh43 and sh45) with validated efficacy .

• Multiple antibody approach: Compare staining patterns using antibodies targeting different epitopes of ZHX2.

• Western blot correlation: Confirm that IHC/IF results correlate with WB findings in the same samples.

• Immunoprecipitation followed by mass spectrometry: For definitive validation, perform IP with the ZHX2 antibody followed by mass spectrometry to confirm target identity.

Several commercial ZHX2 antibodies have undergone knockdown/knockout validation as indicated in their documentation .

How can ZHX2 antibodies be effectively used in ChIP and ChIP-seq experiments?

For chromatin immunoprecipitation of ZHX2:

• Crosslinking: Use 1% formaldehyde for 10 minutes at room temperature for most cell types.

• Chromatin fragmentation: Sonicate to generate fragments of 200-500 bp.

• Antibody selection: Use ChIP-validated ZHX2 antibodies (like 20136-1-AP) . Polyclonal antibodies often perform better in ChIP due to their recognition of multiple epitopes.

• Input amount: 2-5 μg of antibody per 25-100 μg of chromatin.

• Controls: Include:

  • IgG control matched to host species of ZHX2 antibody

  • Input chromatin (non-immunoprecipitated)

  • Positive control region (known ZHX2 binding sites)

• Analysis approaches:

  • For targeted analysis: qPCR of specific promoter regions (e.g., YTHDF2 promoter in liver cells )

  • For genome-wide analysis: Next-generation sequencing of ChIP DNA

ZHX2 ChIP-seq studies have revealed:

• In ccRCC: 75% overlap with p65-binding motifs and strong enrichment for H3K4me3 and H3K27ac marks

• In TNBC: Co-occupancy with HIF1α at transcriptionally active promoters

• In macrophages: Significant overlap with Jun and Bcl6 binding sites

These binding patterns reflect ZHX2's context-dependent roles in different cell types.

What approaches can be used to study ZHX2 protein-protein interactions?

Multiple approaches can be employed to investigate ZHX2 protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use 1-4 μg of ZHX2 antibody per 500-1000 μg of protein lysate

  • Validated interactions include: NF-YA, ZHX1, HIF1α, RelA/p65, aminopeptidase A, and EPHRIN B1

  • Use mild lysis buffers (e.g., NP-40 buffer) to preserve protein-protein interactions

• Proximity ligation assay (PLA):

  • Detects protein interactions in situ in fixed cells/tissues

  • Requires antibodies from different host species for ZHX2 and its potential binding partner

• Bimolecular fluorescence complementation (BiFC):

  • Fusion of ZHX2 and potential interacting protein with complementary fragments of a fluorescent protein

  • Requires molecular cloning expertise and expression vector systems

• Pull-down assays with tagged ZHX2:

  • Several studies have successfully used FLAG-HA-tagged ZHX2 constructs

  • Can be combined with mass spectrometry for unbiased interactome analysis

• Mammalian two-hybrid system:

  • Useful for mapping specific domains involved in interactions

  • Has been used to characterize ZHX2 interactions with other family members

For studying ZHX2 homodimerization or heterodimerization with ZHX1, use crosslinking approaches to stabilize these interactions before immunoprecipitation .

How can researchers effectively use ZHX2 antibodies to study its role in ferroptosis and diabetes-induced liver injury?

Recent research has revealed ZHX2's critical role in regulating ferroptosis during diabetes-induced liver injury. To investigate this:

• Expression analysis in diabetic models:

  • Western blot/IHC analysis of ZHX2 levels in:

    • High-fat diet (HFD) and streptozotocin (STZ)-induced diabetic mice

    • High glucose (HG)-treated hepatic cells (e.g., Huh7)

  • Compare with levels of YTHDF2, GPX4, and SLC7A11

• Overexpression/knockdown studies:

  • Create stable ZHX2 overexpression and knockdown hepatic cell lines

  • Measure ferroptotic markers:

    • Lipid peroxidation (BODIPY 581/591 C11 staining)

    • Iron accumulation (Prussian blue staining)

    • Glutathione levels

    • Expression of ferroptosis regulators (GPX4, SLC7A11)

  • Challenge cells with ferroptosis inducers (e.g., erastin, RSL3)

  • Assess rescue with ferroptosis inhibitors (e.g., ferrostatin-1)

• Mechanistic studies:

  • ChIP assays using ZHX2 antibodies to examine binding to YTHDF2 promoter

  • RNA immunoprecipitation to study YTHDF2 binding to m6A-modified ZHX2 mRNA

  • Luciferase reporter assays to study transcriptional regulation

• In vivo verification:

  • Analyze liver sections from control and ZHX2-overexpressing diabetic mice

  • Perform dual immunofluorescence for ZHX2 and ferroptosis markers

The mechanistic pathway involves ZHX2 inhibiting YTHDF2 transcription, while YTHDF2 promotes ZHX2 mRNA degradation, forming a regulatory feedback loop that influences ferroptosis in diabetes-induced liver injury .

What are common issues with ZHX2 detection and how can they be addressed?

Researchers may encounter several challenges when working with ZHX2 antibodies:

For challenging tissues or cells with low ZHX2 expression, signal amplification systems such as tyramide signal amplification can significantly improve detection limits .

How can researchers optimize co-localization studies involving ZHX2?

For effective co-localization studies with ZHX2 and its interacting partners:

• Antibody compatibility:

  • Choose primary antibodies from different host species (e.g., rabbit anti-ZHX2 and mouse anti-partner protein)

  • Ensure antibodies have been validated for IF/ICC applications

• Sample preparation optimization:

  • For nuclear proteins: Use 0.5% Triton X-100 permeabilization (10 minutes)

  • Fix with 4% paraformaldehyde (10-15 minutes) to preserve protein-protein interactions

  • Perform antigen retrieval if necessary (especially for tissue sections)

• Staining protocol adjustments:

  • Sequential staining may be preferable to simultaneous incubation

  • Include extensive washing steps (4-5 washes, 5 minutes each)

  • Use highly cross-adsorbed secondary antibodies to prevent cross-reactivity

• Imaging considerations:

  • Use confocal microscopy for accurate co-localization assessment

  • Acquire Z-stacks to evaluate 3D co-localization

  • Set proper thresholds to minimize bleed-through between channels

• Quantitative analysis:

  • Calculate Pearson's or Mander's correlation coefficients

  • Perform intensity correlation analysis

  • Use specialized co-localization software (e.g., JACoP plugin for ImageJ)

Key co-localization partners to investigate include:

• NF-κB p65 in ccRCC cells

• HIF1α in TNBC cells

• Aminopeptidase A in podocyte cell membrane

• EPHRIN B1 in slit diaphragm

How should researchers interpret contradictory findings regarding ZHX2's role in different cancer types?

ZHX2 exhibits context-dependent roles that can appear contradictory across different cancer types and cellular contexts:

• Tumor suppressor vs. oncogene dichotomy:

  • Functions as tumor suppressor in:

    • Hepatocellular carcinoma (HCC)

    • Lymphoma

    • Myeloma

  • Functions as oncogene in:

    • Clear cell renal carcinoma (ccRCC)

    • Triple-negative breast cancer (TNBC)

    • Multiple osteosarcoma

    • Gastric cancer

• Methodological considerations for resolving contradictions:

  • Cell type specificity: Use multiple cell lines from the same tissue source

  • Expression level analysis: Quantify ZHX2 expression across cancer stages using both mRNA and protein detection

  • Functional validation: Perform both overexpression and knockdown experiments

  • Pathway analysis: Determine which specific pathways are affected by ZHX2 in each context

  • Interactome differences: Identify tissue-specific binding partners using IP-MS

  • Mouse models: Develop tissue-specific conditional knockout models

• Molecular mechanisms explaining context-dependence:

  • In HCC: ZHX2 represses oncofetal genes like AFP and GPC3

  • In ccRCC: ZHX2 activates NF-κB signaling and MEK/ERK pathway

  • In TNBC: ZHX2 functions as HIF1α cofactor to promote oncogenic signaling

  • In NK cells: ZHX2 restricts maturation and function through IL-15 response regulation

• Research approach recommendations:

  • Focus on context-specific molecular mechanisms rather than general ZHX2 function

  • Consider post-translational modifications in different tissues

  • Analyze specific DNA-binding residues (Arg491, Arg581, Arg674) important for function

  • Examine interaction with tissue-specific transcription factors

This context-dependent behavior makes ZHX2 a complex but promising target for tissue-specific therapeutic interventions.

What emerging techniques could enhance ZHX2 research beyond traditional antibody applications?

Several cutting-edge approaches can advance ZHX2 research beyond conventional antibody applications:

• CRISPR/Cas9 genome editing:

  • Tagged endogenous ZHX2 (e.g., FLAG, GFP knock-in) for live cell imaging and ChIP-seq

  • Domain-specific mutations to study the role of specific zinc finger or homeodomain regions

  • Creation of inducible ZHX2 knockout models for temporal control

• Single-cell technologies:

  • scRNA-seq combined with computational approaches to identify cell-specific ZHX2 regulatory networks

  • Single-cell ATAC-seq to understand chromatin accessibility changes mediated by ZHX2

  • Single-cell proteomics to analyze cell-specific ZHX2 interactomes

• Advanced imaging techniques:

  • Super-resolution microscopy (STORM, PALM) to visualize ZHX2 nuclear organization

  • Live-cell imaging with tagged ZHX2 to monitor dynamic responses to stimuli

  • FRAP (Fluorescence Recovery After Photobleaching) to study ZHX2 mobility and binding kinetics

• Genomics and epigenomics integration:

  • CUT&RUN or CUT&Tag as alternatives to traditional ChIP with improved sensitivity

  • HiChIP to study 3D genomic interactions involving ZHX2-bound regions

  • Integration of ZHX2 binding data with histone modification landscapes

• Structural biology approaches:

  • Cryo-EM to resolve ZHX2 protein complexes

  • Hydrogen-deuterium exchange mass spectrometry to map protein interaction interfaces

  • In silico modeling based on the three arginine residues (Arg491, Arg581, Arg674) identified as critical for DNA binding

These advanced methods can provide deeper mechanistic insights into ZHX2's multifaceted roles across different biological contexts.

How can researchers effectively design studies to explore the therapeutic potential of targeting ZHX2?

To investigate ZHX2 as a therapeutic target, researchers should consider these methodological approaches:

• Identification of disease-specific mechanisms:

  • In diabetes-associated liver injury: Focus on the ZHX2-YTHDF2-ferroptosis axis

  • In TNBC: Target ZHX2-HIF1α interaction and downstream pathways

  • In ccRCC: Focus on ZHX2-mediated NF-κB activation

  • In immune disorders: Target ZHX2's role in NK cell maturation

• Therapeutic strategy development:

  • Small molecule inhibitors: Screen for compounds that disrupt key ZHX2 interactions

  • Peptide inhibitors: Design peptides targeting critical residues (Arg491, Arg581, Arg674)

  • siRNA/antisense approaches: Develop tissue-specific delivery systems

  • Proteolysis-targeting chimeras (PROTACs): Design molecules promoting ZHX2 degradation

• Preclinical validation methods:

  • Patient-derived xenografts to test efficacy of ZHX2-targeting strategies

  • Organoid systems for drug screening in physiologically relevant contexts

  • Conditional knockout mouse models to validate target safety

  • Combination therapy approaches with established treatments

• Biomarker development:

  • Develop assays for ZHX2 expression/activity as patient stratification tools

  • Identify downstream gene signatures that predict response to ZHX2 targeting

  • Create phospho-specific antibodies for activated ZHX2 detection

  • Design companion diagnostics for clinical trials

• Translational considerations:

  • For NK cell-based immunotherapy: ZHX2-deficient NK cells showed enhanced antitumor activity

  • For diabetes complications: ZHX2 overexpression rescued diabetes-induced liver injury

  • For TNBC: Consider small molecules disrupting ZHX2-HIF1α interaction

  • For renal carcinoma: Target ZHX2-mediated Sunitinib resistance mechanisms

These approaches can guide the development of context-specific therapeutic strategies targeting ZHX2 in various disease states.

What bioinformatic approaches can researchers use to analyze ZHX2 ChIP-seq data in conjunction with transcriptomic data?

To maximize insights from integrated analysis of ZHX2 binding and gene expression:

• Data generation and preprocessing:

  • Generate paired ChIP-seq and RNA-seq data from the same biological samples

  • Process ChIP-seq data using standard pipelines (e.g., MACS2 for peak calling)

  • Normalize RNA-seq data appropriately (e.g., DESeq2, edgeR)

• Integrative analysis workflow:

  • Assign ChIP-seq peaks to nearest genes or by proximity within defined windows

  • Correlate peak strength with gene expression levels

  • Identify direct ZHX2 targets (showing both binding and expression changes)

  • Compare binding patterns across conditions (e.g., normal vs. disease state)

• Advanced computational approaches:

  • Motif enrichment analysis to identify co-factors (ZHX2 shows significant overlap with p65 and HIF1α motifs)

  • Gene set enrichment analysis (GSEA) of ZHX2-bound genes

  • Network analysis to identify key hubs in ZHX2 regulatory networks

  • Integration with public ChIP-seq datasets (e.g., ENCODE, Roadmap Epigenomics)

• Visualization strategies:

  • Generate heatmaps of ZHX2 binding around transcription start sites

  • Create genome browser tracks showing ZHX2 binding with gene expression data

  • Use circular plots to visualize long-range interactions

• Validation approaches:

  • Luciferase reporter assays for selected target promoters

  • Site-directed mutagenesis of ZHX2 binding sites

  • eQTL analysis to associate ZHX2 binding with genetic variation

Published studies have revealed context-specific binding patterns:

• In ccRCC: 75% overlap between ZHX2 and p65 binding sites, enriched for H3K4me3 and H3K27ac

• In TNBC: Co-occupancy of ZHX2 and HIF1α at active promoters

• In macrophages: Overlap with apoptosis regulators Jun and Bcl6

How can researchers effectively design studies to investigate the ZHX2-YTHDF2-ferroptosis regulatory axis in metabolic diseases?

To comprehensively investigate the newly identified ZHX2-YTHDF2-ferroptosis axis:

• Experimental model selection:

  • In vivo models:

    • High-fat diet (HFD) and streptozotocin (STZ)-induced diabetic mice

    • Liver-specific ZHX2 knockout or overexpression mice

    • Genetic models of obesity (ob/ob, db/db)

  • In vitro models:

    • Hepatocyte cell lines (Huh7, HepG2) exposed to high glucose

    • Primary hepatocytes from diabetic models

    • 3D liver organoids for physiologically relevant studies

• Comprehensive analysis approach:

  • Expression profiling:

    • Measure ZHX2, YTHDF2, GPX4, SLC7A11 at protein and mRNA levels

    • Assess correlation with disease severity markers

  • Molecular mechanism investigation:

    • ChIP assays to study ZHX2 binding to YTHDF2 promoter

    • RNA immunoprecipitation to examine YTHDF2 binding to m6A-modified ZHX2 mRNA

    • m6A-seq to map global m6A modifications in diabetic conditions

  • Ferroptosis assessment:

    • Lipid peroxidation markers (MDA, 4-HNE, BODIPY 581/591 C11)

    • Iron concentration and distribution

    • GSH/GSSG ratio measurement

    • Cell viability assays with ferroptosis inducers/inhibitors

• Therapeutic intervention studies:

  • ZHX2 overexpression using viral vectors in diabetic models

  • YTHDF2 inhibition approaches

  • Ferroptosis inhibitors (ferrostatin-1, liproxstatin-1)

  • Combination approaches targeting multiple pathway components

• Translational aspects:

  • Analysis of human diabetic liver samples for the pathway components

  • Identification of potential biomarkers for pathway activation

  • Screening of compounds that modulate this axis

This systematic approach can uncover the therapeutic potential of targeting the ZHX2-YTHDF2-ferroptosis axis in metabolic liver diseases and potentially other conditions where ferroptosis plays a pathological role .

What are important considerations for validating and reporting ZHX2 antibody specificity in publications?

Proper validation and reporting of ZHX2 antibody specificity is critical for research reproducibility:

• Essential validation experiments:

  • Western blot showing a single band at the expected molecular weight (92-100 kDa)

  • ZHX2 knockdown/knockout controls showing reduced/absent signal

  • Immunoprecipitation followed by mass spectrometry confirmation

  • Comparison of results using multiple antibodies targeting different epitopes

• Comprehensive antibody reporting:

  • Manufacturer and catalog number

  • Clone name/number for monoclonal antibodies

  • Lot number (particularly important for polyclonal antibodies)

  • Host species and antibody type

  • Epitope information (if available)

  • RRID (Research Resource Identifier) for unambiguous identification

• Experimental details to include:

  • Complete protocol with all buffer compositions

  • Antibody concentration/dilution used

  • Incubation conditions (time, temperature)

  • Detection method details

  • Image acquisition parameters

  • Any image processing performed

• Controls documentation:

  • Positive controls (tissues/cells known to express ZHX2)

  • Negative controls (antibody omission, isotype controls)

  • Peptide competition controls where applicable

  • Secondary-only controls for background assessment

• Ethical considerations:

  • Address potential conflicts of interest related to antibody manufacturers

  • Consider data sharing of full-length blots/images

  • Discuss limitations of the antibodies used

Following these guidelines ensures transparency and reproducibility in ZHX2 research and aligns with the growing emphasis on antibody validation in the scientific community.

How should researchers interpret and reconcile contradictory results when studying ZHX2 in different biological contexts?

When faced with contradictory findings about ZHX2 function:

• Systematic approach to contradictions:

  • Context mapping: Clearly define the cellular/tissue context of each finding

  • Variable isolation: Identify specific experimental variables that might contribute to differences

  • Model system comparison: Consider inherent differences between in vitro, in vivo, and clinical samples

  • Temporal dynamics: Assess whether differences reflect time-dependent processes

• Biological mechanisms explaining contradictions:

  • Cell type-specific cofactors: ZHX2 interacts with different partners (HIF1α in TNBC, p65 in ccRCC)

  • Posttranslational modifications: Different tissues may modify ZHX2 differently

  • Genetic background: Consider strain differences in animal models

  • Disease stage effects: ZHX2's role may change during disease progression

• Technical considerations in data interpretation:

  • Antibody differences: Confirm that different studies are detecting the same form of ZHX2

  • Knockdown/overexpression levels: Consider dose-dependent effects

  • Off-target effects: Critically evaluate genetic manipulation approaches

  • Statistical power: Assess whether sample sizes are sufficient

• Reconciliation strategies:

  • Design experiments that directly test context-dependency hypotheses

  • Perform side-by-side comparisons in multiple systems

  • Use consistent methodology across contexts for direct comparison

  • Consider developing computational models that integrate conflicting data

• Reporting guidelines:

  • Transparently discuss contradictions with existing literature

  • Avoid overgeneralizing findings to contexts not directly studied

  • Consider publishing negative or contradictory results

  • Suggest specific hypotheses to explain contextual differences