NKRF Antibody

Basic Research Questions

• What is NKRF and what are its key cellular functions?

NKRF (NF-κB repressing factor) is a multifunctional stress-regulated protein that plays essential roles in nucleolar homeostasis and cell survival under proteotoxic stress conditions. Unlike conventional heat shock proteins that prevent protein misfolding and aggregation, NKRF acts as an unconventional stress protein with critical functions in:

• Maintaining proper ribosomal RNA (rRNA) processing

• Preventing accumulation of aberrant rRNA precursors and discarded fragments

• Regulating nucleolar-nucleoplasmic trafficking during stress responses

• Inhibiting transcription of specific genes through binding to NRE (negative regulatory element) sequences

• Modulating inflammatory responses through interactions with the NF-κB pathway

NKRF has been identified as a HSF1 target gene, highlighting its importance in cellular stress adaptation mechanisms. During heat stress, NKRF acts as a thermosensor, translocating from the nucleolus to the nucleoplasm, with nucleolar pools being replenished during recovery through HSF1-mediated NKRF resynthesis .

• How do NKRF antibodies help in studying cellular stress responses?

NKRF antibodies provide valuable tools for investigating various aspects of cellular stress responses through multiple methodological approaches:

Researchers can use NKRF antibodies to monitor changes in NKRF localization, expression, and interaction partners during various stress conditions, providing insights into cellular adaptation mechanisms. For example, immunofluorescence staining has confirmed co-localization of NKRF with p50 in cellular studies, while ChIP analysis has demonstrated NKRF binding to specific promoter regions containing NRE sequences .

• What validation methods should be used to verify NKRF antibody specificity?

Proper validation of NKRF antibody specificity is crucial for ensuring experimental reliability. Researchers should implement the following methodological approaches:

• Knockout/knockdown controls: Compare antibody signals between wild-type cells and those with NKRF knockdown or knockout. For example, validation could employ the NKRF-specific siRNAs that have shown high knockdown efficiency (e.g., SiR-Nkrf-3: 5'-CCTGTAGCAACCAACATGT-3') .

• Peptide competition assays: Pre-incubate the antibody with excess purified NKRF peptide before application to samples; specific signals should be blocked.

• Multiple antibody validation: Use at least two different NKRF antibodies targeting distinct epitopes to confirm consistent results.

• Positive control samples: Include samples known to express NKRF (e.g., cardiac fibroblasts or heat-stressed cells) as positive controls.

• Western blot analysis: Verify that the antibody detects a band of the expected molecular weight (~77 kDa for human NKRF).

These validation steps are particularly important when studying NKRF, as its expression levels change dynamically under different stress conditions, potentially leading to variable detection sensitivity .

• What are optimal sample preparation techniques for NKRF immunodetection?

Effective sample preparation is critical for successful NKRF immunodetection in various experimental contexts:

For Immunohistochemistry/Immunofluorescence:

• Fixation: 4% paraformaldehyde for 10-15 minutes at room temperature preserves NKRF structure while maintaining epitope accessibility

• Permeabilization: 0.1% Triton X-100 for 5-10 minutes enables antibody access to intracellular compartments

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) may enhance detection of nuclear NKRF

• Blocking: 5% BSA or normal serum for 1 hour at room temperature reduces background staining

For Western Blotting:

• Lysis buffer composition: Use RIPA buffer supplemented with protease inhibitors, phosphatase inhibitors, and DNase I

• Subcellular fractionation: For studies examining NKRF translocation, separate nuclear/nucleolar fractions from cytoplasmic components

• Sample denaturation: Heat samples at 95°C for 5 minutes in Laemmli buffer containing DTT or β-mercaptoethanol

• Gel percentage: 10% SDS-PAGE gels provide optimal resolution for NKRF

For Chromatin Immunoprecipitation:

• Crosslinking: 1% formaldehyde for 10 minutes at room temperature

• Sonication: Optimize to achieve DNA fragments of 200-500 bp

• Pre-clearing: Use protein A/G beads to reduce non-specific binding

• Antibody concentration: Typically 2-5 μg per ChIP reaction

• How can NKRF antibodies be used to study inflammatory signaling pathways?

NKRF antibodies provide valuable tools for investigating inflammatory signaling mechanisms, particularly through the NF-κB pathway, using the following methodological approaches:

Co-Immunoprecipitation Studies:

• Use NKRF antibodies to pull down protein complexes and analyze interactions with NF-κB components (particularly p50)

• Recommended protocol: Lyse cells in non-denaturing buffer, pre-clear with protein A/G beads, incubate with NKRF antibody overnight at 4°C, capture with fresh beads, wash stringently, and analyze by western blotting for NF-κB components

• Research has demonstrated that NKRF interacts with p50 but not p65 in total cardiac fibroblasts

Chromatin Immunoprecipitation:

• Apply NKRF antibodies to identify genomic binding sites containing NRE sequences

• Focus on promoter regions of inflammatory genes or those involved in stress responses

• For example, NKRF has been shown to bind to the NRE sequence (AATTCCTGA) in the HuR promoter at positions -1493 to -1485 upstream of the transcription start site

Sequential ChIP (Re-ChIP):

• Perform initial ChIP with NKRF antibody followed by a second immunoprecipitation with antibodies against NF-κB components

• This approach can identify genomic regions co-occupied by both NKRF and NF-κB factors

Functional Validation:

• Combine NKRF antibody-based detection methods with NKRF overexpression or knockdown experiments

• For example, studies have shown that NKRF overexpression hinders p65 and p50 binding to the HuR promoter in TNF-α-treated cardiac fibroblasts

Advanced Research Questions

• How can researchers optimize ChIP protocols for NKRF binding studies?

Chromatin immunoprecipitation (ChIP) with NKRF antibodies requires specific optimization strategies to ensure successful identification of NKRF binding sites:

Protocol Optimization for NKRF ChIP:

• Crosslinking Optimization:

  • Standard formaldehyde crosslinking (1% for 10 minutes) may be insufficient for detecting transient NKRF-DNA interactions

  • Consider dual crosslinking with DSG (disuccinimidyl glutarate, 2 mM) for 30 minutes followed by formaldehyde

  • For nucleolar proteins like NKRF, extended crosslinking times (15-20 minutes) may improve recovery

• Chromatin Fragmentation:

  • Sonication conditions should be carefully optimized to achieve fragments of 200-300 bp

  • For nucleolar chromatin, increased sonication power or duration may be necessary

  • Verify fragmentation efficiency by agarose gel electrophoresis before proceeding

• Antibody Selection and Validation:

  • Test multiple NKRF antibodies recognizing different epitopes

  • Validate antibody specificity through Western blotting of nuclear extracts

  • Determine optimal antibody concentration through titration experiments (typically 2-5 μg)

• Negative Controls:

  • Include IgG-only immunoprecipitation as negative control

  • Consider using samples from NKRF-knockdown cells as additional controls

  • For example, research has shown no enrichment in IgG lanes when examining NKRF binding to the HuR promoter

• Primer Design for Target Validation:

  • Design primers flanking potential NRE sites (AATTCCTCTGA or similar sequences)

  • Include primers for known NKRF targets as positive controls

  • Design primers with amplicon sizes of 80-200 bp for optimal qPCR detection

  • For studying potential NKRF binding to new targets, design multiple primer pairs covering the promoter region (as demonstrated in studies examining Mmp2 and Mmp9 promoters)

• What are the methodological considerations when using NKRF antibodies to study stress-induced nucleolar dynamics?

Investigating stress-induced nucleolar dynamics with NKRF antibodies requires specialized approaches:

Experimental Design Considerations:

• Stress Induction Protocols:

  • Heat shock: 42°C for 1 hour followed by recovery at 37°C

  • Proteotoxic stress: Proteasome inhibitors (MG132, 10 μM for 4-6 hours)

  • Oxidative stress: Hydrogen peroxide (0.5 mM for 30-60 minutes)

  • TNF-α treatment: 10 ng/mL in serum-free medium

• Time-Course Analysis:

  • Collect samples at multiple timepoints during stress and recovery phases

  • For example: baseline, 15 min, 30 min, 1 hour, 2 hours, 4 hours, and 8 hours post-stress

  • This approach can capture the dynamics of NKRF nucleolar-nucleoplasmic trafficking

• Subcellular Fractionation:

  • Separate nucleolar, nucleoplasmic, and cytoplasmic fractions

  • Verify fraction purity using markers (nucleolin for nucleoli, lamin B for nuclear matrix)

  • Quantify NKRF levels in each fraction by Western blotting

• Live-Cell Imaging:

  • For real-time analysis, consider using cells expressing fluorescently-tagged NKRF

  • Combine with NKRF antibody validation in fixed cells to confirm proper localization

• Co-localization Studies:

  • Use dual immunofluorescence with NKRF antibodies and markers for:

    • Nucleolar components (fibrillarin, nucleolin)

    • Processing bodies (DCP1, GW182)

    • Stress granules (G3BP, TIA-1)

  • Quantify co-localization using appropriate algorithms and statistical analysis

Research has demonstrated that NKRF acts as a thermosensor, translocating from the nucleolus to the nucleoplasm during heat stress, with nucleolar pools being replenished during recovery .

• How can NKRF antibodies be used to investigate rRNA processing mechanisms?

NKRF antibodies can provide valuable insights into rRNA processing mechanisms through several methodological approaches:

RNA-Protein Interaction Studies:

• RNA Immunoprecipitation (RIP):

  • Use NKRF antibodies to immunoprecipitate NKRF-RNA complexes

  • Extract and analyze associated RNAs through RT-PCR or RNA sequencing

  • Focus on rRNA precursors and processing intermediates

  • Compare RIP results between normal and stress conditions

• Crosslinking Immunoprecipitation (CLIP):

  • UV-crosslink RNA-protein complexes before immunoprecipitation with NKRF antibodies

  • This technique provides higher specificity for direct RNA-protein interactions

  • Sequence recovered RNAs to identify NKRF binding sites on rRNA precursors

• Nucleolar Run-On Assays:

  • Combine with NKRF immunofluorescence to correlate rRNA synthesis with NKRF localization

  • Compare results between control and NKRF-depleted cells

• Pulse-Chase Analysis of rRNA Processing:

  • Label nascent rRNAs with 5-EU (5-ethynyl uridine)

  • Track processing kinetics in control versus NKRF-depleted cells

  • Use NKRF antibodies for simultaneous immunofluorescence detection

• Analysis of rRNA Processing Defects:

  • Use Northern blotting to detect accumulation of aberrant rRNA precursors

  • Compare processing patterns between normal cells and those with NKRF knockdown

  • Research has shown that NKRF is crucial for correct rRNA processing and preventing accumulation of aberrant rRNA precursors and discarded fragments

• What experimental approaches can reveal NKRF's role in the NF-κB signaling pathway?

To thoroughly investigate NKRF's role in NF-κB signaling, researchers should consider these methodological approaches:

Interaction and Functional Studies:

• Co-Immunoprecipitation Experiments:

  • Use NKRF antibodies to pull down protein complexes followed by Western blotting for NF-κB components

  • Perform reciprocal Co-IP using antibodies against NF-κB subunits (p50, p65)

  • Compare interaction profiles under basal and stimulated conditions (e.g., TNF-α treatment)

  • Research has demonstrated that NKRF interacts with p50 but not p65 in total cardiac fibroblasts through Co-IP analysis

• Chromatin Occupancy Analysis:

  • Perform sequential ChIP (Re-ChIP) to identify genomic regions co-occupied by NKRF and NF-κB components

  • Compare chromatin occupancy patterns before and after inflammatory stimulation

  • Research has shown that NKRF overexpression hinders p65 and p50 binding to target promoters, such as the HuR promoter in TNF-α-treated cells

• Transcriptional Regulation Studies:

  • Use luciferase reporter assays with wild-type and NRE-deleted promoters

  • Combine with NKRF overexpression or knockdown to assess functional impact

  • For example, NKRF significantly inhibited the activity of firefly luciferase driven by the wild-type HuR promoter, but this effect disappeared when the NRE sequence was deleted

• NF-κB Activation Dynamics:

  • Track nuclear translocation of p65/p50 in control versus NKRF-overexpressing cells

  • Quantify phosphorylation of IκB and p65 by Western blotting

  • Measure DNA binding activity using electrophoretic mobility shift assays (EMSA)

• Pathway Inhibition Studies:

  • Use NF-κB pathway inhibitors (e.g., IMD 0354) to determine whether NKRF effects are dependent on NF-κB activity

  • Research has shown that the negative transcriptional regulation of HuR by NKRF requires an active NF-κB pathway

• How can researchers apply NKRF antibodies in cardiac fibroblast research models?

Recent research has identified important roles for NKRF in cardiac fibroblasts, with implications for cardiac remodeling and dysfunction. Here are methodological approaches for applying NKRF antibodies in this research context:

Cardiac Fibroblast Research Applications:

• Primary Cardiac Fibroblast Isolation and Culture:

  • Isolate cardiac fibroblasts from ventricular tissue using collagenase digestion

  • Use NKRF antibodies to confirm expression levels across different passages

  • Consider studying passages 1-3 as optimal for maintaining native characteristics

• NKRF Expression Analysis During Cardiac Stress:

  • Apply stimuli relevant to cardiac pathology:

    • TNF-α (10 ng/mL) for inflammatory stress

    • Angiotensin II (100 nM) for hypertrophic stimulus

    • Hypoxia (1% O₂) for ischemic conditions

  • Track NKRF expression and localization changes using antibody-based detection methods

  • Research has shown that NKRF mRNA levels significantly decrease after TNF-α induction in cardiac fibroblasts

• Functional Impact Assessment:

  • Combine NKRF antibody detection with functional assays:

    • Migration assays (scratch wound, Boyden chamber)

    • Collagen production (Sirius Red staining, hydroxyproline assay)

    • MMP activity (gelatin zymography, fluorogenic substrate assays)

  • Research has demonstrated that NKRF overexpression inhibits cardiac fibroblast migration and invasion by downregulating MMP2 and MMP9 expression and activities

• In Vivo Validation Approaches:

  • Use cardiac fibroblast-specific NKRF knockout (NKRF-CKO) mouse models

  • Apply adeno-associated viruses (AAVs) encoding NKRF for in vivo overexpression

  • Use NKRF antibodies for immunohistochemical analysis of cardiac tissue sections

  • Studies have employed S100a4-Cre strain crossed with NKRF flox/flox mice to generate cardiac fibroblast-specific NKRF knockout models

• Translation to Human Pathology:

  • Apply NKRF antibodies to human cardiac tissue samples

  • Compare NKRF expression patterns between normal and pathological specimens

  • Correlate with markers of inflammation and fibrosis