NKAPL Antibody

What is NKAPL and what are its biological functions in cellular processes?

NKAPL (NFKB activating protein like) is a nuclear protein that functions as a transcriptional regulator with multiple critical biological roles:

• Transcriptional repression: NKAPL acts as a transcriptional repressor, particularly in Notch signaling pathways .

• Role in development: NKAPL is essential for T-cell development, where it functions as a transcriptional corepressor of Notch-mediated signaling .

• Spermatogenesis regulation: NKAPL is robustly expressed in differentiating spermatogonia and early spermatocytes, and is essential for spermatogenesis. Deletion of NKAPL in mice causes complete arrest at the level of pachytene spermatocytes .

• Transcription elongation control: Recent research has identified NKAPL as a factor that facilitates RNA polymerase II pause-release and bridges transcription elongation with initiation by binding to promoter-associated nascent transcripts .

• R-loop interaction: NKAPL co-localizes with DNA-RNA hybrid R-loop structures at GAA-rich loci to enhance R-loop formation and facilitate Pol II pause-release .

• NF-κB activation: Though primarily a repressor, NKAPL can weakly activate NF-κB in a dose-dependent manner .

What is the tissue expression pattern of NKAPL, and how does it differ from its related gene NKAP?

NKAPL shows a highly restricted tissue expression pattern compared to its related gene NKAP:

NKAPL Expression:

• Highly tissue-specific, predominantly expressed in testis

• Expression is developmentally regulated in testis:

  • Low expression until 2 weeks of age

  • Significantly upregulated (approximately 50-fold) from 3 weeks onward

  • Correlates with appearance of pachytene spermatocytes and early round spermatids

NKAP Expression:

• Ubiquitously expressed across various tissues and cell types

• Essential for normal development in multiple cell lineages

Evolutionary relationship:

• NKAPL is an autosomal gene that lacks introns

• It appears to have originated as a retrotransposed gene from the X-linked NKAP

• This retrotransposition event occurred before the divergence of eutherians and metatherians

What applications are NKAPL antibodies most commonly used for in research?

NKAPL antibodies are employed in various experimental techniques to study the protein's expression, localization, and function:

Both commercial polyclonal and recombinant antibodies are available, with rabbit being the most common host species for NKAPL antibody production .

What are the best practices for validating NKAPL antibody specificity in experimental applications?

Validation of NKAPL antibody specificity is crucial for reliable research outcomes. Based on the latest recommendations from the scientific community, the following approaches represent best practices:

Gold standard: Genetic knockout validation approach

• Use of parental and NKAPL knockout cell lines for side-by-side comparison represents the most rigorous validation method

• This approach has demonstrated superior results compared to orthogonal validation methods, particularly for immunofluorescence applications (80% confirmation rate vs. 38% for orthogonal methods)

Recommended validation protocol:

• Western blot validation:

  • Use positive control samples with known NKAPL expression (HeLa, Jurkat, K-562, or A549 cells)

  • Include negative control (NKAPL knockout cells if available)

  • Expect bands at 47-51 kDa; be aware that observed molecular weight may be higher than predicted (44.86 kDa)

• Immunohistochemistry validation:

  • Test using paraffin-embedded tissues with known NKAPL expression

  • Include appropriate controls (tissue from NKAPL knockout mice)

  • Optimize antibody concentration (typically 1:50-1:200 dilution)

• Immunofluorescence validation:

  • The mosaic approach is recommended: image a mixture of wild-type and knockout cells in the same visual field to reduce imaging and analysis biases

  • Look for nuclear localization (NKAPL contains nuclear localization signals at amino acid sequences GSQKRRRFSE, HSTKKKRKKK, and KPSKRKHKKYY)

• Recombinant protein controls:

  • Test antibody against recombinant NKAPL protein

  • Include related proteins (e.g., NKAP) to confirm specificity

How does NKAPL function in transcription regulation, particularly with respect to RNA Pol II pause-release?

Recent research has revealed NKAPL as a critical factor in transcription elongation with specific mechanistic details:

NKAPL's role in transcription regulation:

• RNA Pol II pause-release facilitation:

  • NKAPL depletion prolongs RNA Pol II pauses at promoter-proximal regions

  • NKAPL knockout increases RNA Pol II traveling ratio (ratio of Pol II density in promoter-proximal region to gene body), indicating increased Pol II pausing

  • NKAPL binding signals overlap with promoter-proximal Pol II peaks

• R-loop interaction mechanism:

  • NKAPL binds to promoter-associated nascent transcripts

  • Co-localizes with DNA-RNA hybrid R-loop structures at GAA-rich loci

  • Enhances R-loop formation which facilitates Pol II pause-release

  • eCLIP-seq analysis showed that 46% of NKAPL RNA binding peaks are located within promoter regions (±2 kb from TSS)

• Integration with transcription initiation:

  • NKAPL depletion stalls the SOX30/HDAC3 transcription initiation complex on chromatin

  • Functions as a bridge between transcription elongation and initiation processes

• Gene expression impact:

  • In NKAPL knockout mice, majority of affected genes show downregulation rather than upregulation

  • Most significantly affected are haploid gene expression during spermatogenesis

Molecular interaction mechanisms:

• NKAPL contains an arginine/serine-rich (RS) domain with RNA-binding capacity

• Metagene analysis revealed highest NKAPL binding at approximately 40 nt downstream of transcription start sites, corresponding to known hotspots for Pol II pause release

What genetic and molecular associations exist between NKAPL and human diseases?

NKAPL has been implicated in several human diseases through genetic variants and molecular mechanisms:

Schizophrenia associations:

• Genetic evidence:

  • The NKAPL locus has been identified as a risk locus for schizophrenia, particularly in Han Chinese populations

  • The rs1635 variant (c.455C>A, resulting in amino acid change T152N) has been specifically associated with cognitive function in early-onset schizophrenia (EOS)

• Functional impact:

  • Patients with the CC genotype (encoding NKAPL-152T) performed better in cognitive domains of speed processing, trail making test, and category fluency compared to those with AA or AC genotypes

  • The peripheral blood mRNA expression level of NKAPL in NKAPL-152N carriers is significantly lower than in NKAPL-152T carriers

  • The phosphorylation level of NKAPL-152N is significantly decreased compared to NKAPL-152T, potentially affecting protein function

Rheumatoid Arthritis:

• Fine-mapping analyses identified six NKAPL locus variants in a single haplotype block showing association with rheumatoid arthritis

• Among these SNPs, rs35656932 in the zinc finger 193 gene and rs13208096 in the NKAPL gene remained significant after conditional logistic regression

• These associations remained significant after conditioning on SNPs tagging the HLA-shared epitope (SE) DRB1*0401 allele

Male infertility:

• NKAPL depletion causes male infertility in mice by blocking meiotic exit and downregulating haploid genes

• Genetic variants in NKAPL are associated with azoospermia in humans

• Mice carrying an NKAPL frameshift mutation (M349fs) show defective meiotic exit

What are the technical considerations for using NKAPL antibodies in immunohistochemistry and immunofluorescence applications?

Immunohistochemistry technical considerations:

• Tissue fixation and preparation:

  • Validated for paraffin-embedded tissues

  • Antigen retrieval may be necessary due to epitope masking during fixation

• Optimal antibody dilutions:

  • Typical working dilutions range from 1:50 to 1:200 for IHC-P

  • Specific recommendations from validated studies:

    • 1:500 dilution used successfully for rat hind brain tissue

    • 1:500 dilution used successfully for mouse duodenum tissue

• Controls and validation:

  • Positive controls: Tissues with known NKAPL expression (testis, particularly from post-pubertal animals)

  • Negative controls: Tissues from NKAPL knockout animals when available

Immunofluorescence technical considerations:

• Cell preparation:

  • Fixation: Standard 4% paraformaldehyde fixation suitable for nuclear proteins

  • Permeabilization: Required due to nuclear localization of NKAPL

• Antibody concentration:

  • Recommended concentrations: 0.25-2 μg/mL

  • Validation using transfected cells expressing tagged NKAPL is recommended

• Expected localization pattern:

  • Primarily nuclear localization due to nuclear localization signals

  • Transfection experiments with NKAPL-inserted EGFP-expression vector confirm nuclear localization

• Co-localization studies:

  • Consider co-staining with R-loop markers as NKAPL co-localizes with R-loop structures

  • Co-staining with RNA Pol II can help identify sites of transcriptional regulation

• Advanced validation approach:

  • Mosaic method: Mix wild-type and knockout cells in the same field of view

  • This reduces imaging and analysis biases and provides internal negative controls

How does NKAPL protein interact with the Notch signaling pathway and what experimental approaches can be used to study this?

NKAPL functions as a transcriptional corepressor in the Notch signaling pathway, with important implications for development and disease:

NKAPL's role in Notch signaling:

• Transcriptional repression mechanism:

  • Acts as a transcriptional repressor of Notch-mediated signaling

  • Associates with chromatin at Notch-regulated promoters, such as the SKP2 promoter

  • Required for T-cell development through its role in Notch signaling

• Protein complex formation:

  • Associates with several molecules of the Notch corepressor complex:

    • CIR (CBF1 interacting corepressor)

    • HDAC3 (Histone deacetylase 3)

    • CSL (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1)

Experimental approaches to study NKAPL-Notch interactions:

• Protein-protein interaction studies:

  • Co-immunoprecipitation (Co-IP): Can be used to verify interactions between NKAPL and Notch corepressor complex components

  • Example approach: Insert full-length mouse Cir, Hdac3, Csl, and NICD of Notch1-3 by PCR from mouse testis cDNA, tag with FLAG, and use for Co-IP experiments

• Transcriptional activity assays:

  • Luciferase reporter assays: Use CSL binding consensus sequence (GTGGGAA×4) and SV40 promoter sequence in reporter constructs

  • Example approach: Generate pEluc-CSLs by inserting CSL binding consensus sequence and SV40 promoter sequence into pEluc vector

• Chromatin binding analysis:

  • ChIP-seq: Determine genome-wide binding sites of NKAPL at Notch-regulated genes

  • ChIP-qPCR: Validate binding to specific Notch target gene promoters

• Genetic perturbation studies:

  • NKAPL knockout or knockdown followed by gene expression analysis of Notch target genes

  • NKAPL overexpression studies to observe effects on Notch signaling components

  • Study shows NKAPL overexpression in germline stem cells induces changes in stem cell markers and reduces differentiation factors through the Notch signaling pathway

• Domain-specific analysis:

  • The DUF926 domain at the C-terminus is conserved between NKAP and NKAPL and may be critical for function

  • Structure-function studies using domain deletion or mutation can reveal specific regions required for Notch interaction

What are the observed discrepancies between predicted and observed molecular weights of NKAPL protein, and what factors might explain these differences?

The NKAPL protein consistently shows higher apparent molecular weight in SDS-PAGE than predicted from its amino acid sequence, which presents important considerations for researchers:

Observed discrepancies:

• Predicted molecular weight: 44.86 kDa based on the 1188 base pair open reading frame encoding NKAPL

• Observed molecular weight in immunoblotting:

  • 52 kDa in mouse testis lysates

  • 47-50 kDa in human cell lines

  • 47 kDa, 51 kDa observed in HeLa whole cell and nuclear extracts

Potential factors explaining these discrepancies:

• Post-translational modifications:

  • Phosphorylation: NKAPL is subject to threonine phosphorylation, as demonstrated by immunoprecipitation using phosphothreonine antibodies

  • Different phosphorylation states may contribute to observed size variations

  • The genetic variant rs1635 (T152N) affects phosphorylation levels, with NKAPL-152N showing decreased phosphorylation

• Alternative splicing or initiation:

  • Although NKAPL is intronless (being a retrotransposed gene), alternative transcription start sites could yield different protein variants

• Protein-specific characteristics:

  • The arginine/serine-rich domain and other charged regions may affect protein mobility in SDS-PAGE

  • Nuclear localization signals (GSQKRRRFSE at the 15th, HSTKKKRKKK at 180th, and KPSKRKHKKYY at 189th amino acid positions) contain charged residues that can influence migration

• Technical considerations:

  • Different SDS-PAGE systems used across studies (e.g., 10% vs. 12% gels)

  • Variations in sample preparation and running conditions

Recommendations for researchers:

• When using Western blot to detect NKAPL, be prepared to observe bands at ~47-52 kDa rather than at the predicted 44.86 kDa

• Include positive controls with known NKAPL expression (e.g., testis tissue for mouse studies, HeLa cells for human studies)

• Consider running recombinant NKAPL protein alongside samples when available

• When performing immunoblotting after immunoprecipitation, account for potential shifts due to post-translational modifications

What methodological approaches can be used to study NKAPL RNA-binding properties and its role in R-loop biology?

NKAPL has emerged as an important RNA-binding protein that interacts with R-loops. The following methodological approaches can be employed to investigate these properties:

RNA-binding characterization techniques:

• Enhanced Crosslinking and Immunoprecipitation (eCLIP-seq):

  • Successfully used to profile RNA targets of NKAPL on chromatin

  • Protocol fundamentals:

    • UV crosslinking to stabilize protein-RNA interactions

    • Immunoprecipitation with validated NKAPL antibodies

    • Library preparation and high-throughput sequencing

  • Key findings:

    • Over 99.5% of NKAPL eCLIP peaks located within protein-coding transcripts

    • 46% of peaks located within promoter regions (±2 kb from TSS)

    • Highest binding at ~40 nt downstream of transcription start sites

• RNA Electrophoretic Mobility Shift Assay (EMSA):

  • Can be used to evaluate direct binding of purified NKAPL to RNA sequences

  • Useful for determining sequence specificity and binding affinity

  • RNA containing tandem GAA repeats would be primary candidates based on existing data

• RNA Immunoprecipitation (RIP):

  • Less stringent than CLIP but useful for initial characterization

  • Can be coupled with qRT-PCR to validate binding to specific transcripts

R-loop biology investigation methods:

• DNA-RNA Immunoprecipitation (DRIP):

  • Uses the S9.6 antibody that specifically recognizes DNA-RNA hybrids

  • Can be coupled with sequencing (DRIP-seq) to map R-loops genome-wide

  • Can assess how NKAPL depletion affects R-loop formation and stability

• R-loop visualization techniques:

  • Immunofluorescence with S9.6 antibody to visualize R-loops

  • Co-localization studies with NKAPL antibodies to confirm spatial association

• RNase H sensitivity assays:

  • RNase H specifically degrades RNA in DNA-RNA hybrids

  • Can be used to confirm R-loop identity and study how NKAPL protects or enhances R-loop stability

• Functional assays:

  • RNA Polymerase II traveling ratio analysis:

    • Calculate the relative ratio of Pol II read density in the promoter-proximal region to the gene body

    • NKAPL depletion leads to increased traveling ratios, indicating more paused Pol II

  • Nascent RNA analysis (e.g., GRO-seq, PRO-seq) to measure effects on transcription elongation

• Structural studies:

  • The RS domain (arginine/serine-rich) in NKAPL is likely involved in RNA binding

  • Domain-specific mutations can identify critical residues for RNA binding and R-loop interaction

Experimental considerations:

• Tissue/cell selection is important as NKAPL is predominantly expressed in testis, specifically in spermatogonia and early spermatocytes

• For studies in other cell types, consider overexpression systems with appropriate controls

• RNA targets to prioritize include promoter-proximal transcripts, particularly those containing GAA-rich sequences