NKX2-4 Antibody

What is NKX2-4 and what is its normal biological function?

NKX2-4 (NK2 Homeobox 4) is a member of the NK-2 homeobox family of transcription factors involved in embryonic development and cell fate determination. It functions as a transcription factor with sequence-specific DNA binding capabilities . In normal development, NKX2-4 plays a role in ventral forebrain development, particularly in the hypothalamus and preoptic region . Studies in zebrafish have demonstrated that nkx2.1 and nkx2.4 genes function partially redundantly during development . NKX2-4 is closely related to NKX2-1 and is specifically localized to the ventral diencephalon .

Where is NKX2-4 typically expressed during development and in adult tissues?

NKX2-4 exhibits a highly restricted expression pattern:

During embryonic development, NKX2-4 is detected in the posterior hypothalamus and later in the head region . In adult organisms, expression becomes even more restricted, with detection primarily in the testis . Analyses of public RNA-seq datasets have confirmed the absence of NKX2-4 expression in developing and mature hematopoietic cells while confirming its presence in the hypothalamus, pituitary gland, and testis . Interestingly, NKX2-4 is not represented in standard expression profiling arrays, which may explain why its transcriptional deregulation has not been widely reported in research literature .

What criteria should be used to select an appropriate NKX2-4 antibody?

When selecting an NKX2-4 antibody, researchers should consider multiple factors to ensure experimental success:

Most commercially available NKX2-4 antibodies are rabbit polyclonal antibodies that react with human and mouse samples . For example, ab189202 is a rabbit polyclonal antibody suitable for ICC, WB, IHC-P, and ICC/IF applications with mouse and human samples . Some antibodies are specifically designed not to cross-react with other NK2 homeobox family members, which is important for specificity .

How should I validate the specificity of an NKX2-4 antibody?

Thorough validation of NKX2-4 antibodies requires multiple complementary approaches:

• Positive and negative controls: Test the antibody on tissues known to express NKX2-4 (testis, hypothalamus) and tissues known not to express it (most adult tissues) .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application. This should eliminate specific signals, as demonstrated in Western blot validation of ab189202 using A20 cell lysate with immunizing peptide .

• Knockdown validation: Compare antibody staining in wild-type cells versus cells where NKX2-4 has been knocked down using siRNA. This approach has been successfully implemented in AML cell line OCI-M2 .

• Multiple antibody comparison: Use antibodies that recognize different epitopes of NKX2-4 and compare their staining patterns. Available antibodies target various regions including N-terminal, C-terminal, and internal regions .

• Molecular weight verification: Confirm that the antibody detects a band of the expected molecular weight (approximately 36 kDa for NKX2-4) in Western blot applications .

What are the recommended positive and negative control samples for NKX2-4 antibody testing?

For rigorous validation of NKX2-4 antibodies, the following controls are recommended:

Positive controls:

• Adult testis tissue (confirmed NKX2-4 expression)

• Hypothalamus and pituitary gland tissues

• AML cell line OCI-M2 (high NKX2-4 expression)

• B-cell lymphoma cell line U-2932 (elevated NKX2-4 levels)

• A20 cells (validated for immunocytochemistry applications)

Negative controls:

• Most adult tissues (due to highly restricted expression)

• Normal hematopoietic cells (demonstrated absence of NKX2-4 expression)

• Most AML cell lines (exceptions being OCI-M2 and low-level in THP-1)

For immunohistochemical applications, formalin-fixed, paraffin-embedded human skin tissue has been successfully used with antibodies like ab189202 at 5 μg/ml concentration .

Western Blotting (WB)

• Recommended concentration: 1 μg/ml for antibodies like ab189202

• Expected molecular weight: 36 kDa

• Sample preparation: Standard cell lysis with protease inhibitors

• Controls: Include cell lines with known NKX2-4 expression (A20, OCI-M2)

Immunohistochemistry (IHC-P)

• Recommended concentration: 5 μg/ml for formalin-fixed, paraffin-embedded tissues

• Antigen retrieval: Heat-induced epitope retrieval with citrate buffer (pH 6.0)

• Detection system: ABC (avidin-biotin complex) or polymer-based systems

• Controls: Include testis tissue as positive control

Immunofluorescence (IF)/Immunocytochemistry (ICC)

• Recommended concentration: 2.5-20 μg/ml depending on the antibody

• Fixation: 4% paraformaldehyde for 10-15 minutes

• Permeabilization: 0.1-0.3% Triton X-100 to access nuclear proteins

• Nuclear counterstain: DAPI or Hoechst recommended

• Note: NKX2-4 protein has been observed in both nucleus and cytoplasm

How can I determine the subcellular localization of NKX2-4?

NKX2-4 can exhibit both nuclear and cytoplasmic localization, suggesting regulation via nuclear import mechanisms . For accurate subcellular localization studies:

• Immunofluorescence approach:

  • Fix cells appropriately (4% paraformaldehyde)

  • Permeabilize with 0.1-0.3% Triton X-100

  • Use validated NKX2-4 antibody (ab189202 has been used at 20 μg/ml)

  • Include markers for specific cellular compartments (nuclear envelope, nucleoli)

  • Employ confocal microscopy for precise localization

• Nuclear-cytoplasmic fractionation:

  • Separate nuclear and cytoplasmic fractions using established protocols

  • Analyze fractions by Western blot with NKX2-4 antibody

  • Include markers for nuclear (e.g., Lamin B) and cytoplasmic (e.g., GAPDH) fractions

• Cell models:

  • A20 cells have been successfully used for immunofluorescent analysis of NKX2-4

  • OCI-M2 cells show high NKX2-4 expression and can serve as a model system

Analyses of NKX2-4 in OCI-M2 cells revealed distribution in both nuclear and cytoplasmic compartments, suggesting functional regulation via nuclear-cytoplasmic shuttling mechanisms .

What approaches can I use to study NKX2-4 transcriptional activity?

To investigate NKX2-4's function as a transcription factor:

• Reporter gene assays:

  • Construct reporters containing putative NKX2-4 binding sites upstream of a minimal promoter

  • Co-transfect with NKX2-4 expression vectors in appropriate cell models

  • Measure reporter activity under various conditions

  • Include binding site mutations as specificity controls

• Target gene expression analysis:

  • Perform siRNA-mediated knockdown of NKX2-4

  • Analyze expression changes in potential target genes via RT-qPCR

  • Established targets include ETV2, HEY1, FOXA1, MAML2, SIX5 (activated) and FLI1, SIRPA (repressed)

• Chromatin immunoprecipitation (ChIP):

  • Use validated NKX2-4 antibodies for immunoprecipitation

  • Design primers for potential binding sites in target genes

  • For genome-wide binding analysis, perform ChIP-seq

  • Integrate with expression data to identify direct targets

• Transcription factor network analysis:

  • Investigate cooperative binding with other transcription factors

  • Assess NKX2-4 within developmental transcription factor networks

  • NKX2-4 forms a mutually activating network with ETV2 and HEY1 in AML cells

What is the role of NKX2-4 in acute myeloid leukemia (AML)?

NKX2-4 has been identified as aberrantly expressed in specific AML subtypes, with significant pathological implications:

In normal hematopoietic cells, NKX2-4 is not expressed, making its presence in AML cells an aberrant feature . The ETV2 locus at 19p13 is often genomically amplified in AML, potentially driving aberrant NKX2-4 expression . Repression of FLI1 by NKX2-4 is particularly significant as FLI1 is a master factor for myelopoiesis that drives megakaryocytic differentiation and suppresses erythroid differentiation .

Comparative analysis of clinical samples has confirmed that key regulators and target genes of NKX2-4 identified in the OCI-M2 cell line model are similarly expressed in AML-M6 (acute erythroblastic leukemia) patients, validating the clinical relevance of these findings .

How does NKX2-4 interact with other transcription factors in regulatory networks?

NKX2-4 participates in complex transcription factor networks that impact both normal development and pathological conditions:

Upstream regulators of NKX2-4:

• SOX7 binds to a SOX consensus site in the NKX2-4 promoter region

• IRF6 binds at -2648 bp, ETV2 at -2021 bp, and HEY1 within exon 2 of NKX2-4

• In AML cell line OCI-M2, these endothelial transcription factors aberrantly activate NKX2-4

• In Xenopus, the homeodomain protein XANF is necessary for NKX2-4 activation

NKX2-4 downstream targets:

• NKX2-4 activates ETV2 and HEY1, forming a mutually activating network in AML cells

• NKX2-4 activates SIX5 and FOXA1 while repressing FLI1, a master factor for myelopoiesis

• These regulatory interactions impact myeloid differentiation processes

Comparison with related NKL homeobox genes:

• NKX2-3, another aberrantly expressed NKL homeobox gene in AML, activates FLI1, contrasting with NKX2-4's repression of FLI1

• This differential regulation may explain how different NKL homeobox genes contribute to specific AML subtypes

• Systematic analysis has identified 18 deregulated NKL homeobox genes in AML

What experimental approaches can be used to identify NKX2-4 target genes?

Multiple complementary approaches are recommended for comprehensive identification of NKX2-4 target genes:

• Expression profiling after gene manipulation:

  • Perform siRNA-mediated knockdown of NKX2-4 followed by RNA-seq or microarray analysis

  • Compare expression profiles before and after knockdown to identify differentially expressed genes

  • This approach identified several NKX2-4 targets in OCI-M2 cells, including transcription factors and signaling molecules

  • Gene set annotation analysis can reveal biological processes regulated by NKX2-4

• Bioinformatic promoter analysis:

  • Screen for potential NKX2-4 binding sites in regulatory regions of candidate genes

  • Use tools like UCSC genome browser and CIS-BP database to identify consensus sites

  • This approach identified potential NKX2-4 binding sites in ETV2 (-1235 bp) and HEY1 (-961 bp)

• Direct binding assessment:

  • Perform chromatin immunoprecipitation (ChIP) with validated NKX2-4 antibodies

  • Design reporter gene assays with wild-type and mutated binding sites to confirm direct regulation

  • Reporter gene assays confirmed direct regulation of NKX2-4 by ETV2

• Validation by targeted gene expression analysis:

  • Select candidate target genes from profiling data for validation

  • Perform RT-qPCR to confirm expression changes after NKX2-4 knockdown

  • This approach confirmed that NKX2-4 activates FOXA1, MAML2, and SIX5 and represses FLI1 and SIRPA in OCI-M2 cells

• Clinical correlation:

  • Compare findings from cell line models with primary patient samples

  • Analyze public datasets from patient samples to validate clinical relevance

  • This approach showed that key NKX2-4 targets identified in OCI-M2 were similarly expressed in AML-M6 patients

What approaches are effective for NKX2-4 knockdown studies?

For studying NKX2-4 function through loss-of-function approaches:

• siRNA-mediated knockdown:

  • Design and validate specific siRNAs targeting NKX2-4 mRNA

  • Optimize transfection conditions for your cell model

  • Verify knockdown efficiency at both mRNA level (RT-qPCR) and protein level (Western blot)

  • Include appropriate controls (non-targeting siRNA)

  • This approach has been successfully used in OCI-M2 cells

• CRISPR-Cas9 gene editing:

  • Design guide RNAs targeting the NKX2-4 gene

  • Generate knockout cell lines or animal models

  • Verify knockout by sequencing and expression analysis

  • This approach allows for complete elimination of NKX2-4 rather than partial knockdown

• Morpholino-based knockdown (for developmental studies):

  • Design morpholinos targeting NKX2-4 splice junctions or translation start site

  • Verify specificity using GFP-reporter constructs containing morpholino target sequences

  • This approach has been used for studying nkx2.4 function in zebrafish development

• Phenotypic analysis:

  • Assess effects on cell proliferation, apoptosis, and differentiation

  • Analyze expression changes in potential target genes

  • In OCI-M2 cells, NKX2-4 knockdown did not significantly impact proliferation or apoptosis but affected expression of several target genes

How can I analyze NKX2-4 DNA binding specificity?

To analyze NKX2-4 binding to target DNA sequences:

• Chromatin Immunoprecipitation (ChIP):

  • Use validated NKX2-4 antibodies for immunoprecipitation

  • Perform ChIP-qPCR to examine binding at specific loci

  • For genome-wide binding analysis, perform ChIP-seq

  • Include appropriate controls (IgG, input DNA)

• Electrophoretic Mobility Shift Assay (EMSA):

  • Design labeled oligonucleotides containing predicted NKX2-4 binding sites

  • Use recombinant NKX2-4 protein or nuclear extracts from cells expressing NKX2-4

  • Include competition assays with unlabeled oligonucleotides

  • Perform supershift assays with NKX2-4 antibodies to confirm specificity

• DNA-Protein Interaction ELISA:

  • Immobilize biotinylated DNA oligonucleotides containing NKX2-4 binding sites

  • Incubate with recombinant NKX2-4 or nuclear extracts

  • Detect binding using NKX2-4 antibodies

  • This provides a quantitative measure of binding efficiency

• Reporter Gene Assays:

  • Clone predicted binding sites upstream of a minimal promoter and reporter gene

  • Perform reporter assays in cells with or without NKX2-4 expression

  • Include mutated binding sites as controls

  • This approach has been used to confirm ETV2 regulation of NKX2-4

What considerations are important when studying NKX2-4 in developmental contexts?

For developmental studies of NKX2-4:

• Model system selection:

  • Consider zebrafish models, which have been used to study nkx2.4 function

  • Mouse models are appropriate for mammalian development studies

  • Cell differentiation models can recapitulate aspects of development in vitro

• Spatiotemporal expression analysis:

  • Characterize normal NKX2-4 expression patterns throughout development

  • Use in situ hybridization to map mRNA expression

  • Complement with immunohistochemistry for protein localization

  • NKX2-4 is expressed in the posterior hypothalamus and head during embryonic development

• Functional redundancy considerations:

  • NKX2.1 and NKX2.4 genes can function partially redundantly during development

  • Design experiments to account for potential compensatory mechanisms

  • Consider combined knockdown approaches

  • In zebrafish studies, knockdown of nkx2.1, nkx2.4a, and nkx2.4b revealed redundant functions

• Loss-of-function approaches:

  • Use morpholinos or CRISPR-Cas9 for targeted gene knockdown/knockout

  • Verify knockdown efficiency and specificity

  • When using morpholinos, validate specificity with appropriate controls including GFP-reporter constructs

• Gain-of-function approaches:

  • Use controlled expression systems rather than broad overexpression

  • Note that broad overexpression of NKX2-4 can cause severely abnormal development

  • Consider tissue-specific or inducible expression systems