Phospho-NFKB1 (Ser893) Antibody

What is the specificity of Phospho-NFKB1 (Ser893) antibody?

Phospho-NFKB1 (Ser893) antibody specifically detects endogenous levels of NF-kappa-B p105 protein only when phosphorylated at the serine 893 residue. This antibody is typically produced against synthesized peptides derived from human NF-kappaB p105/p50 around the phosphorylation site of Ser893, specifically in the amino acid range of 860-909 . The specificity of this antibody is critical for researchers studying phosphorylation-dependent regulation of NF-kappa-B signaling pathways. Validation methods typically involve using phosphatase treatments or comparing signals between phosphorylated and non-phosphorylated protein samples.

To confirm specificity in your experiments, consider:

• Including both phosphorylated and non-phosphorylated controls

• Using competing peptides to validate binding specificity

• Employing phosphatase treatments to demonstrate phospho-specificity

What are the recommended applications for Phospho-NFKB1 (Ser893) antibody?

Phospho-NFKB1 (Ser893) antibody is suitable for multiple research applications including:

These applications enable researchers to study the phosphorylation state of NFKB1 in various experimental contexts . When planning experiments, it's advisable to optimize dilutions based on your specific experimental system, including cell type, tissue origin, and detection method.

How should Phospho-NFKB1 (Ser893) antibody be stored and handled?

For optimal stability and performance, Phospho-NFKB1 (Ser893) antibody should be stored at -20°C for up to one year from the date of receipt . The antibody is typically supplied in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide, which helps maintain stability during freeze-thaw cycles .

Best practices for handling:

• Aliquot upon first thaw to minimize freeze-thaw cycles

• Avoid repeated freeze-thaw cycles as they can lead to protein denaturation and loss of activity

• When thawing, allow the antibody to reach room temperature completely before use

• Centrifuge the vial briefly before opening to collect all liquid at the bottom

• Follow manufacturer's recommendations for short-term storage (2-8°C) if the antibody will be used within a week

What is the NFKB1 protein and its role in cellular signaling?

NF-kappa-B (NFKB1) is a pleiotropic transcription factor present in almost all cell types and functions as the endpoint of various signal transduction events . The protein exists in two forms:

• p105: the precursor form

• p50: the mature, processed form

NFKB1 plays crucial roles in numerous biological processes:

• Inflammation and immune response regulation

• Cell differentiation and development

• Cellular growth control

• Tumorigenesis

• Apoptosis regulation

The protein typically forms homo- or heterodimeric complexes with other Rel-like domain-containing proteins including RELA/p65, RELB, REL, and NFKB2/p52, with the p65-p50 heterodimer being the most abundant form . These dimers bind to kappa-B sites in the DNA of target genes, regulating their transcription. Phosphorylation at Ser893 is one of several post-translational modifications that regulate NFKB1 function, particularly affecting its protein interactions, nuclear translocation, and transcriptional activity.

How does phosphorylation at Ser893 affect NFKB1 function compared to other phosphorylation sites?

Phosphorylation of NFKB1 at Ser893 represents one of several critical regulatory modifications that modulate this transcription factor's activity. Unlike phosphorylation at sites such as Ser337 (which affects DNA binding) or Ser927 (which influences processing from p105 to p50), Ser893 phosphorylation appears to primarily regulate protein-protein interactions.

Comparative effects of different NFKB1 phosphorylation sites:

Experimental approaches to study these differences include:

• Site-directed mutagenesis (S893A or S893E) to mimic non-phosphorylated or constitutively phosphorylated states

• Comparative phosphoproteomic analysis

• Kinase inhibitor studies using specific inhibitors for GSK-3β or IKK complex

When designing experiments to investigate Ser893 phosphorylation, researchers should consider the stimulus-specific and cell type-specific nature of this modification, as inflammatory stimuli like TNF-α and IL-1β may differently affect Ser893 phosphorylation compared to other sites.

What are the optimal experimental conditions for detecting Phospho-NFKB1 (Ser893) in Western blot applications?

Detecting Phospho-NFKB1 (Ser893) via Western blot requires careful attention to sample preparation, electrophoresis conditions, and detection protocols. The following methodology has been optimized based on research practices:

Sample Preparation:

• Harvest cells during peak phosphorylation (typically 5-30 minutes after stimulation with TNF-α, LPS, or IL-1β)

• Lyse cells in buffer containing phosphatase inhibitors (10 mM sodium fluoride, 1 mM sodium orthovanadate, 10 mM β-glycerophosphate)

• Maintain samples at 4°C throughout processing to preserve phosphorylation state

Electrophoresis and Transfer:

• Use 8% SDS-PAGE gels to achieve optimal separation of the 105 kDa NFKB1 protein

• Transfer to PVDF membrane at 100V for 90 minutes in cold transfer buffer containing 20% methanol

• Block with 5% BSA (not milk, which contains phosphatases) in TBST for 1 hour

Detection Protocol:

• Incubate with Phospho-NFKB1 (Ser893) antibody at 1:1000 dilution in 5% BSA-TBST overnight at 4°C

• Wash 4 times with TBST, 5 minutes each

• Incubate with HRP-conjugated secondary antibody (anti-rabbit) at 1:5000 for 1 hour at room temperature

• Develop using enhanced chemiluminescence with exposure times of 1-5 minutes

Critical Controls:

• Positive control: Cell lysates treated with TNF-α (20 ng/ml) for 15 minutes

• Negative control: Unstimulated cell lysates

• Specificity control: Lambda phosphatase-treated samples

• Loading control: Total NFKB1 antibody on stripped membrane

For improved signal-to-noise ratio, consider using signal enhancers and longer primary antibody incubation times (up to 24 hours at 4°C).

How can I troubleshoot weak or non-specific signal when using Phospho-NFKB1 (Ser893) antibody?

When encountering weak or non-specific signals with Phospho-NFKB1 (Ser893) antibody, systematic troubleshooting is essential:

Problem: Weak or No Signal

Problem: Non-specific Bands

Advanced Validation Methods:

• Peptide competition assay: Pre-incubate antibody with phospho-peptide immunogen

• Knockout/knockdown validation: Compare signal in NFKB1 knockout or siRNA-treated samples

• Phosphatase treatment: Treat half of your sample with lambda phosphatase

Most researchers find that optimizing the sample preparation is the critical step, particularly ensuring that phosphorylation is preserved throughout the experimental workflow .

What are the differences between various commercial Phospho-NFKB1 (Ser893) antibodies and how does this affect experimental design?

Commercial Phospho-NFKB1 (Ser893) antibodies from different manufacturers exhibit variations that can significantly impact experimental outcomes. Understanding these differences helps in selecting the appropriate antibody for specific applications:

Implications for Experimental Design:

• Species compatibility: Select antibodies validated for your model organism (human, mouse, rat)

• Application specificity: Some antibodies perform better in specific applications (e.g., St John's for WB, Abbexa for IF)

• Epitope considerations: Antibodies recognizing different regions around Ser893 may have different sensitivities to conformational changes

• Validation requirements: Newer antibodies may require more extensive validation in your specific system

When comparing results across studies using different antibodies, consider:

• Standardizing protocols when possible

• Including parallel experiments with different antibodies for critical findings

• Documenting the exact antibody catalog number and lot in publications

How can Phospho-NFKB1 (Ser893) antibody be utilized in multiplexed detection systems?

Multiplexed detection systems allow simultaneous analysis of multiple phosphorylation states or proteins, providing valuable insight into signaling pathway dynamics. Phospho-NFKB1 (Ser893) antibody can be effectively incorporated into these systems through several approaches:

Fluorescence-Based Multiplexing:

• Multi-color immunofluorescence: Combine Phospho-NFKB1 (Ser893) antibody with antibodies against other pathway components (e.g., phospho-IKK, phospho-RelA) using species-specific or isotype-specific secondary antibodies with different fluorophores.

• Proximity Ligation Assay (PLA): Use antibody pairs (phospho-specific and total NFKB1) to detect specific phosphorylation events with spatial resolution. The Abnova antibody pair is specifically designed for this application .

Bead-Based Multiplexing:

• Luminex/Bio-Plex systems: Conjugate Phospho-NFKB1 (Ser893) antibody to spectrally distinct beads for multiplex phosphoprotein analysis.

• CyTOF (mass cytometry): Label with isotope-tagged secondary antibodies for high-dimensional analysis of cell populations.

Sequential Detection Methods:

• Sequential fluorescent Western blotting: Use direct labeled primary antibodies or spectrally distinct secondary antibodies

• Stripping and reprobing: Carefully validate stripping efficiency to ensure complete removal before reprobing

Practical Protocol for Dual Immunofluorescence Detection:

• Fix cells with 4% paraformaldehyde (15 minutes, room temperature)

• Permeabilize with 0.2% Triton X-100 (10 minutes)

• Block with 5% normal serum + 1% BSA (1 hour)

• Incubate with Phospho-NFKB1 (Ser893) rabbit antibody (1:100) and mouse antibody against another target (overnight, 4°C)

• Wash 3× with PBS

• Incubate with anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 594 (1:500, 1 hour)

• Counterstain nuclei with DAPI

• Mount and image using confocal microscopy

When designing multiplexed experiments, consider potential issues with antibody cross-reactivity and implement appropriate controls, including single-antibody staining controls and isotype controls.

What stimuli are most effective for inducing NFKB1 Ser893 phosphorylation?

The phosphorylation of NFKB1 at Ser893 is stimulus-dependent, with different activators showing varying efficiency and kinetics. Understanding these differences is crucial for designing experiments that effectively capture this phosphorylation event:

Methodological Considerations:

• Cell synchronization: Serum-starve cells (0.5-1% serum) for 12-24 hours prior to stimulation to reduce background phosphorylation

• Dose-response assessment: Perform initial titration experiments to determine optimal concentrations for your specific cell type

• Time-course analysis: Capture multiple time points (5, 15, 30, 60, 120 minutes) to identify peak phosphorylation

• Harvesting method: Rapid lysis is critical; direct addition of hot SDS sample buffer can help preserve phosphorylation status

For investigating pathway specificity, consider using appropriate inhibitors as controls:

• IKK inhibitors (BMS-345541, TPCA-1) to block canonical NF-κB activation

• GSK-3β inhibitors (SB216763, CHIR99021) to examine the role of this kinase in Ser893 phosphorylation

• Proteasome inhibitors (MG132, bortezomib) to prevent degradation of IκB and assess effects on NFKB1 phosphorylation

This methodological approach allows researchers to capture the dynamic nature of NFKB1 Ser893 phosphorylation across different experimental contexts.

How should cellular and tissue samples be prepared to preserve NFKB1 Ser893 phosphorylation?

Preserving phosphorylation states during sample preparation is critical for accurate analysis of NFKB1 Ser893 phosphorylation. The following protocols have been optimized to maintain phosphorylation integrity:

Cell Culture Samples:

• Rapid processing: Minimize time between stimulation and lysis (≤1 minute)

• Pre-chilled reagents: Use ice-cold PBS for washing and lysis buffers

• Phosphatase inhibitor cocktail: Include sodium fluoride (10 mM), sodium orthovanadate (1 mM), β-glycerophosphate (10 mM), and phosphatase inhibitor cocktails

• Lysis buffer composition:

  • 50 mM Tris-HCl, pH 7.4

  • 150 mM NaCl

  • 1% NP-40 or Triton X-100

  • 0.5% sodium deoxycholate

  • 0.1% SDS

  • 1 mM EDTA

  • Protease and phosphatase inhibitors (freshly added)

Tissue Samples:

• Flash freezing: Immediately freeze harvested tissues in liquid nitrogen

• Cryosectioning: Maintain frozen state throughout processing

• Homogenization: Use mechanical disruption in the presence of phosphatase inhibitors

• Fixation for IHC/IF: Use 4% paraformaldehyde fixation limited to 15-20 minutes to prevent epitope masking

Critical Parameters for Different Applications:

Validation Method:
To confirm preservation of phosphorylation, process parallel samples with and without phosphatase inhibitors, or treat a portion of your sample with lambda phosphatase before analysis. This provides a direct assessment of phosphorylation state preservation in your specific experimental system .

Can Phospho-NFKB1 (Ser893) antibody be effectively used in CHIP or CHIP-seq applications?

While Phospho-NFKB1 (Ser893) antibodies are primarily validated for Western blot, IHC, IF, and ELISA applications , their use in Chromatin Immunoprecipitation (ChIP) or ChIP-seq requires special considerations and modified protocols. Here's a methodological approach for adapting these antibodies for chromatin studies:

Technical Considerations for ChIP Applications:

• Antibody Validation for ChIP:

  • Test immunoprecipitation efficiency using nuclear extracts before proceeding to chromatin

  • Confirm phospho-specificity in IP formats using phosphatase-treated controls

  • Verify DNA binding capacity of phosphorylated p105/p50 forms in gel shift assays

• Optimized ChIP Protocol:

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

  • Sonication: Optimize to achieve 200-500 bp fragments

  • Pre-clearing: Extended pre-clearing (2 hours) with protein A/G beads

  • Antibody incubation: Higher antibody concentration (5-10 μg per reaction)

  • Increased incubation time: 16-20 hours at 4°C with rotation

  • Stringent washing: Include high salt wash steps to reduce background

• Controls Essential for Validation:

  • Input chromatin (10%)

  • IgG negative control

  • Positive control using antibody against total NFKB1/p50

  • Known NF-κB target gene promoters (e.g., IL-8, IκBα)

• ChIP-qPCR Primer Design:
  Target known NF-κB binding sites in promoters such as:

  • IκBα promoter: Forward 5'-GACGACCCCAATTCAAATCG-3', Reverse 5'-TCAGGCTCGGGGAATTTCC-3'

  • IL-8 promoter: Forward 5'-GGGCCATCAGTTGCAAATC-3', Reverse 5'-TTCCTTCCGGTGGTTTCTTC-3'

• ChIP-seq Considerations:

  • Higher input chromatin amounts (typically double standard ChIP)

  • Sequential ChIP approach may improve specificity

  • Bioinformatic analysis should include motif enrichment for NF-κB binding sites

Biological Interpretation:
It's important to note that phosphorylation at Ser893 may influence DNA binding properties or protein-protein interactions affecting chromatin association. When interpreting ChIP-seq data, researchers should consider that Phospho-NFKB1 (Ser893) might identify a subset of total NFKB1 genomic binding sites, potentially representing specialized functional states of this transcription factor.

While ChIP applications are not explicitly listed in the standard applications for commercial Phospho-NFKB1 (Ser893) antibodies , these methodological adaptations provide a framework for researchers interested in exploring this application.

How should quantitative data from Phospho-NFKB1 (Ser893) immunoblots be normalized and analyzed?

Proper normalization and analysis of Phospho-NFKB1 (Ser893) immunoblot data are essential for accurate interpretation of phosphorylation dynamics. The following methodological approach ensures robust quantification:

Normalization Strategies:

• Dual Detection Approach:

  • Primary method: Normalize phospho-signal to total NFKB1 protein

  • Procedure: Strip and reprobe membrane or use dual-color detection systems

  • Formula: (Phospho-NFKB1 band intensity) ÷ (Total NFKB1 band intensity)

• Loading Control Normalization:

  • Secondary method: Normalize to housekeeping proteins (β-actin, GAPDH, α-tubulin)

  • Note: Less specific than total protein normalization, but useful as secondary verification

• Total Protein Normalization:

  • Alternative approach: Use total protein staining methods (Ponceau S, SYPRO Ruby, Stain-Free gels)

  • Advantage: Addresses limitations of single housekeeping protein references

  • Implementation: Normalize based on total protein content in each lane

Quantification Protocol:

• Capture digital images using a linear detection system (avoid film when possible)

• Analyze band intensities using software (ImageJ, Image Lab, etc.)

• Subtract background from each band

• Calculate phospho/total ratio for each sample

• Normalize experimental conditions to control condition

Statistical Analysis Framework:

Visualization Methods:

• Bar graphs with error bars (SEM or SD) for group comparisons

• Line graphs for time course or dose-response experiments

• Include individual data points along with means for transparent reporting

Interpretative Considerations:

• Transient phosphorylation events may be missed in single time-point analyses

• Signal saturation can mask differences at high expression/phosphorylation levels

• Partial phosphorylation may result in diffuse bands requiring total area quantification

This comprehensive approach to quantification ensures reliable analysis of NFKB1 Ser893 phosphorylation across experimental conditions.

What are the key considerations for interpreting NFKB1 Ser893 phosphorylation in the context of NF-κB signaling pathways?

Interpreting NFKB1 Ser893 phosphorylation requires contextual understanding within the broader NF-κB signaling network. Consider these critical factors when analyzing experimental results:

Pathway Context and Relationship to Other Phosphorylation Events:

• Hierarchical Phosphorylation Relationships:

  • Ser893 phosphorylation often occurs downstream of IKK activation

  • May be influenced by GSK-3β activity in certain cellular contexts

  • Consider sequential phosphorylation events that may precede or follow Ser893 modification

• Functional Integration with Other NF-κB Modifications:

  • Analyze in conjunction with p105 processing to p50

  • Compare with RelA/p65 phosphorylation state (Ser536)

  • Consider IκBα degradation kinetics as context

• Cell Type-Specific Interpretation:

  • Immune cells: Often shows rapid, robust phosphorylation

  • Epithelial cells: May exhibit more sustained, moderate phosphorylation

  • Neurons: Typically demonstrates delayed, prolonged phosphorylation patterns

Functional Significance Framework:

Comparative Analysis Approach:
To fully understand Ser893 phosphorylation, analyze it in parallel with:

• Total NFKB1 protein levels (p105 and p50)

• Nuclear translocation of p50 and p65

• NF-κB transcriptional activity (reporter assays)

• Target gene expression (qPCR or RNA-Seq)

Common Interpretation Pitfalls:

• Assuming phosphorylation always correlates with increased transcriptional activity

• Interpreting phosphorylation without considering potential compensatory mechanisms

• Overlooking the possibility of phosphorylation-dependent protein-protein interactions

• Failing to consider the subcellular localization of phosphorylated NFKB1

How can I design siRNA or CRISPR experiments to validate the specificity of Phospho-NFKB1 (Ser893) antibody signals?

Genetic validation using siRNA knockdown or CRISPR-mediated gene editing provides definitive confirmation of antibody specificity for Phospho-NFKB1 (Ser893). Here's a comprehensive methodology for designing and implementing these validation approaches:

siRNA Knockdown Validation Protocol:

• siRNA Design and Selection:

  • Target multiple regions of NFKB1 mRNA

  • Recommended sequences:

    • siRNA-1: 5'-GGAGACAUCCUUCCGCAAA-3'

    • siRNA-2: 5'-GCAGGUAUUUGACAUAUUA-3'

    • siRNA-3: 5'-GGCUAUAACUCGCCUAGUG-3'

  • Include non-targeting control siRNA with similar GC content

• Transfection Optimization:

  • Cell type-specific optimization of transfection reagent (Lipofectamine RNAiMAX, DharmaFECT)

  • Titrate siRNA concentration (10-50 nM range)

  • Determine optimal time point for maximum knockdown (typically 48-72 hours)

• Validation Experimental Design:

  • Transfect cells with NFKB1 siRNA and control siRNA

  • After optimal knockdown period, stimulate with TNF-α (10 ng/ml for 15 minutes)

  • Prepare lysates for Western blot analysis

  • Probe with both Phospho-NFKB1 (Ser893) and total NFKB1 antibodies

CRISPR/Cas9 Gene Editing Approach:

• gRNA Design for NFKB1 Knockout:

  • Target early exons to ensure complete protein disruption

  • Recommended gRNA sequences:

    • gRNA-1: 5'-GCGGCCTGCACTTCTGACGT-3'

    • gRNA-2: 5'-ACTGTAGTAGCAGAGATGCT-3'

  • Design verification primers to confirm genomic editing

• CRISPR Implementation Strategy:

  • Transient transfection with Cas9 and gRNA plasmids

  • Clonal isolation and screening

  • Genomic verification by sequencing

  • Protein verification by Western blot for total NFKB1

• Ser893 Phospho-Site Mutant Generation:

  • Design HDR template to introduce S893A mutation

  • gRNA targeting Ser893 region: 5'-TCTCAGAGCCCTGAGTTCAA-3'

  • HDR template containing S893A mutation (TCC→GCC)

  • Screen clones by sequencing and verify expression levels

Experimental Controls and Analysis:

Data Interpretation Framework:

• In siRNA experiments: Expect proportional reduction in both total and phospho-specific signal

• In CRISPR knockout: Complete absence of both signals confirms specificity

• In S893A mutants: Loss of phospho-signal with maintained total protein confirms phospho-specificity

This comprehensive genetic validation approach provides the highest level of confidence in the specificity of Phospho-NFKB1 (Ser893) antibody signals and addresses potential concerns about antibody cross-reactivity or off-target binding.

What control experiments should be included when studying NFKB1 Ser893 phosphorylation kinetics?

Robust control experiments are essential for accurate interpretation of NFKB1 Ser893 phosphorylation kinetics. The following methodological framework provides a comprehensive approach to experimental controls:

Essential Control Categories:

• Phosphorylation Specificity Controls:

  • Phosphatase treatment: Treat duplicate samples with lambda phosphatase to confirm phospho-specific signal

  • Competing phosphopeptide: Pre-incubate antibody with phospho-Ser893 peptide to block specific binding

  • Non-phosphorylatable mutant: Compare wild-type to S893A mutant-expressing cells

• Stimulus and Inhibitor Controls:

  • Unstimulated baseline: Establish baseline phosphorylation in resting cells

  • Positive stimulus control: Include TNF-α (10 ng/ml, 15 min) as reference standard

  • Pathway inhibitor controls: IKK inhibitor (BMS-345541, 10 μM) pre-treatment

  • Vehicle controls: Match all solvent conditions for inhibitor studies

• Technical Controls:

  • Loading controls: Total NFKB1 and housekeeping proteins (β-actin, GAPDH)

  • Antibody specificity: Secondary-only controls to assess non-specific binding

  • Cross-reactivity assessment: Validate in NFKB1 knockout/knockdown cells

Comprehensive Control Experiment Panel:

Kinetics Control Methodology:

• Capture appropriate time points: 0, 5, 15, 30, 60, 120, 240 minutes

• Include both rapid and delayed time points: Some phosphorylation events show biphasic patterns

• Maintain consistent stimulus concentration: Prepare master stocks for all time points

• Temperature control: Maintain cells at consistent temperature during stimulation

• Synchronous stimulation and harvest: Use rapid stimulation termination methods

Data Verification Through Complementary Approaches:

• Confirm key findings with alternative antibody from different vendor/clone

• Validate with mass spectrometry phosphoproteomics for critical experiments

• Use phospho-flow cytometry to assess single-cell phosphorylation kinetics

By implementing this comprehensive control framework, researchers can generate highly reliable data on NFKB1 Ser893 phosphorylation kinetics and confidently distinguish biological effects from technical artifacts.

How can Phospho-NFKB1 (Ser893) antibody be used to study disease-relevant NF-κB dysregulation?

Phospho-NFKB1 (Ser893) antibody serves as a valuable tool for investigating disease states characterized by NF-κB pathway dysregulation. The following methodological approach outlines how to effectively employ this antibody in disease-relevant research:

Disease-Specific Research Applications:

• Inflammatory Disorders:

  • Tissue analysis: Compare Ser893 phosphorylation in inflamed vs. healthy tissues

  • Experimental models: Monitor phosphorylation kinetics in:

    • LPS-induced sepsis models

    • DSS-induced colitis

    • Rheumatoid arthritis models

  • Therapeutic evaluation: Assess how anti-inflammatory compounds affect Ser893 phosphorylation

• Cancer Research Applications:

  • Tumor tissue analysis: Compare phosphorylation patterns between tumor and adjacent normal tissue

  • Cell line studies: Analyze constitutive Ser893 phosphorylation in cancer cell lines

  • Correlation studies: Assess relationship between Ser893 phosphorylation and:

    • Tumor grade/stage

    • Therapy resistance

    • Patient outcomes

• Neurodegenerative Diseases:

  • Brain tissue analysis: Examine regional distribution in Alzheimer's/Parkinson's tissues

  • Stimulus-response: Compare neuroinflammatory stimuli responses in disease models

  • Longitudinal studies: Track changes during disease progression

Methodological Considerations by Application:

Translational Research Framework:

• Biomarker development: Assess whether Ser893 phosphorylation levels correlate with disease activity

• Therapy monitoring: Track changes during treatment courses

• Patient stratification: Investigate if baseline phosphorylation predicts treatment response

• Target validation: Use phospho-state as readout for target engagement

Emerging Applications:

• Single-cell analysis: Combine with single-cell techniques to resolve cellular heterogeneity

• Spatial transcriptomics integration: Correlate phosphorylation with local gene expression patterns

• Liquid biopsy development: Explore extracellular vesicle-associated phospho-NFKB1 as biomarker

By applying these methodologies, researchers can leverage Phospho-NFKB1 (Ser893) antibody to gain insights into disease mechanisms, identify potential therapeutic targets, and develop biomarkers for NF-κB pathway dysregulation in various pathological contexts.

What is known about the relationship between NFKB1 Ser893 phosphorylation and other post-translational modifications?

NFKB1 Ser893 phosphorylation exists within a complex network of post-translational modifications (PTMs) that collectively regulate NF-κB signaling. Understanding these interrelationships is crucial for comprehensive pathway analysis:

Interplay with Other Phosphorylation Sites:

• Sequential Phosphorylation Relationships:

  • IKK-mediated phosphorylation at Ser927/932 often precedes Ser893 phosphorylation

  • Ser893 phosphorylation may prime for subsequent modifications at nearby residues

  • GSK-3β-mediated phosphorylation exhibits distinct patterns from IKK-mediated sites

• Functional Coordination:

  • Phosphorylation at Ser337 (DNA binding domain) works in concert with Ser893 to regulate transcriptional activity

  • C-terminal phosphorylation sites (including Ser893) collectively regulate p105 processing and stability

Cross-talk with Other PTM Types:

Methodological Approaches to Study PTM Crosstalk:

• MS/MS-Based Approaches:

  • Phospho-enrichment followed by mass spectrometry

  • Combined PTM enrichment strategies

  • Quantitative MS/MS to track dynamic PTM changes

• Sequential IP Strategies:

  • First IP: Phospho-NFKB1 (Ser893)

  • Second IP: Antibodies against other PTMs (e.g., ubiquitin, SUMO)

  • Analysis: Determine co-occurrence frequency

• Site-Directed Mutagenesis Studies:

  • Generate phospho-mimetic (S893D/E) and phospho-deficient (S893A) mutants

  • Assess impact on other PTMs

  • Create combination mutants to test PTM interdependence

Experimental Design for PTM Crosstalk Analysis:

• Time-course analysis: Track different PTMs after stimulus exposure

• Pharmacological manipulation: Use PTM-specific inhibitors to disrupt specific modifications

• Sequence motif analysis: Identify potential multi-PTM regulatory regions

• Structural analysis: Determine how phosphorylation at Ser893 affects accessibility of other modification sites

This systematic approach to studying PTM relationships provides deeper insight into the complex regulatory network governing NFKB1 function and helps identify key control points in NF-κB signaling that may represent therapeutic targets.