Phospho-NFKB2 (Ser866) Antibody

What is the significance of NF-κB2/p100 phosphorylation at Ser866?

Phosphorylation of NF-κB2/p100 at Ser866 represents a critical regulatory step in the non-canonical NF-κB signaling pathway. This post-translational modification occurs within the degron region of p100 and is essential for targeting the protein for subsequent partial proteasomal degradation, which generates the active p52 transcription factor. The phosphorylation event at Ser866 typically occurs alongside phosphorylation at Ser870, and possibly Ser872, following the accumulation of NF-κB-inducing kinase (NIK) and activation of IκB kinase α (IKKα) . This specific phosphorylation precedes ubiquitination at Lys855, which tags the C-terminal portion of p100 for proteasomal processing . Understanding this phosphorylation is crucial for investigating abnormalities in immune function, as mutations affecting this region have been implicated in immunodeficiency disorders and cytopenias.

How does Phospho-NFKB2 (Ser866) antibody differ from antibodies targeting other phosphorylation sites?

Phospho-NFKB2 (Ser866) antibody specifically recognizes NF-κB2/p100 protein that has been phosphorylated at the serine 866 residue, making it distinct from antibodies targeting other phosphorylation sites. While some commercial antibodies detect dual phosphorylation at Ser866/870 , others are designed to recognize only the Ser866 phosphorylation site . This specificity allows researchers to precisely monitor this particular phosphorylation event without cross-reactivity to non-phosphorylated p100 or p100 phosphorylated at different sites. The antibody serves as a valuable tool for studying the specific kinase-substrate interactions involved in the non-canonical NF-κB pathway activation, particularly through selective detection of this regulatory modification that occurs prior to proteasomal processing.

What are the main research applications for Phospho-NFKB2 (Ser866) antibodies?

Phospho-NFKB2 (Ser866) antibodies serve multiple research applications for investigating the non-canonical NF-κB pathway:

• Western Blotting: These antibodies are commonly used at dilutions of 1:500-1:2,000 to detect phosphorylated p100 in cell and tissue lysates, enabling quantitative assessment of pathway activation .

• Immunoprecipitation: At approximately 1:50 dilution, these antibodies can isolate phosphorylated p100 complexes from cellular extracts for subsequent analysis of interacting proteins .

• Immunohistochemistry: Used at dilutions of 1:50-1:100 to visualize the tissue distribution and subcellular localization of phosphorylated p100 in fixed specimens .

• Immunofluorescence: Applied at dilutions of 1:50-1:100 for high-resolution imaging of phosphorylated p100 in cultured cells, often combined with other markers to study pathway dynamics .

These applications collectively enable researchers to track the activation status of the non-canonical NF-κB pathway across diverse experimental contexts and disease models.

How do mutations in the phosphorylation sites of NFKB2 affect protein function and disease pathogenesis?

Mutations affecting the phosphorylation sites of NFKB2, particularly those around Ser866, fundamentally disrupt the regulated processing of p100 to p52, leading to significant pathological consequences. Research has identified that heterozygous mutations that truncate the NF-κB2 precursor protein proximal to the degron region enable signal-independent production of p52, resulting in immunodeficiency or cytopenias in affected patients . Mouse models carrying mutations in this region, such as the S866fs variant, demonstrate altered p100/p52 ratios, suggesting disrupted homeostasis in p100 processing .

A particularly informative case involved a patient with a de novo T-nucleotide insertion at c.2598 of NFKB2, creating a frameshift mutation (p.A867Cfs*) adjacent to the Ser866 phosphorylation site . This mutation was associated with combined immune deficiency, indicating that aberrant NFKB2 signaling not only affects humoral immunity but also interferes with TCR-mediated T cell proliferation . These findings collectively demonstrate that precise phosphorylation at Ser866 and nearby residues maintains a critical threshold of p100 degradation that, when disrupted, leads to immune dysregulation and associated clinical manifestations.

What is the relationship between NIK activation, IKKα recruitment, and p100 Ser866 phosphorylation in the non-canonical NF-κB pathway?

The relationship between NIK activation, IKKα recruitment, and p100 Ser866 phosphorylation represents a precisely coordinated sequence in the non-canonical NF-κB pathway. Upon stimulation by specific ligands (such as BAFF, CD40L, or lymphotoxin-β), NIK protein stabilizes intracellularly, escaping its normal degradation fate . This accumulated NIK then phosphorylates and activates IKKα, which subsequently phosphorylates p100 at specific serine residues, crucially including Ser866 and Ser870 .

This phosphorylation event occurs within the p100 degron region and serves as a molecular tag that recruits the SCFβ-TrCP E3 ubiquitin ligase complex. The complex then catalyzes the ubiquitination of Lys855, marking the C-terminal portion of p100 for selective proteasomal degradation . This partial proteolysis generates p52, which forms transcriptionally active dimers primarily with RelB. The entire cascade exemplifies a tightly regulated signaling mechanism where each step—NIK stabilization, IKKα activation, and sequential p100 phosphorylation—ensures both signal specificity and appropriate magnitude of pathway activation.

How do p100/p52 ratios reflect the efficiency of phosphorylation-dependent processing in different experimental models?

The p100/p52 ratio serves as a critical indicator of the efficiency of phosphorylation-dependent processing in various experimental models. Research with genetically modified mice carrying different NFKB2 variants demonstrates that this ratio reliably reflects the resistance of the p100 protein pool to processing into p52 . In wild-type conditions, this ratio maintains homeostatic balance, while perturbations in phosphorylation sites alter this equilibrium.

For example, studies of the S866fs NFKB2 variant revealed that heterozygous mice (+/S866fs) showed decreased levels of both p100 and p52, whereas homozygous mice (S866fs/S866fs) exhibited halved p100 abundance with relatively normal p52 levels . This peculiar phenomenon suggests that mutations affecting Ser866 phosphorylation create distinct thresholds for p100 degradation that define lymphoid tissue development and function.

The experimental data reveals a pattern where:

These variable ratios across models demonstrate that phosphorylation-dependent processing efficiency directly impacts immune system development and function, with specific threshold effects defining the transition between normal and pathological states .

What are the optimal sample preparation techniques for detecting phosphorylated Ser866 in NF-κB2/p100?

Detection of phosphorylated Ser866 in NF-κB2/p100 requires meticulous attention to sample preparation to preserve the labile phospho-epitope while minimizing background interference. The following protocol incorporates critical considerations:

• Cell Lysis Buffer Composition: Use a phospho-preserving lysis buffer containing:

  • 20 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • 1 mM EDTA

  • 1 mM EGTA

  • Phosphatase inhibitors (10 mM NaF, 1 mM Na3VO4, 1 mM β-glycerophosphate)

  • Protease inhibitor cocktail

• Temperature Control: Perform all extraction procedures at 4°C to minimize phosphatase activity.

• Quick Processing: Process samples immediately after collection, as phosphorylation states can rapidly change.

• Cell Stimulation: For optimal detection, stimulate cells with non-canonical NF-κB pathway activators (BAFF, CD40L, etc.) for 6-8 hours before lysis .

• Protein Quantification: Use Bradford or BCA assays that are compatible with phosphatase inhibitors.

• Sample Storage: Store lysates in single-use aliquots at -80°C with phosphatase inhibitors to prevent freeze-thaw cycles that may degrade phospho-epitopes.

When running Western blots, load 20-50 μg of protein per lane and include appropriate controls, such as lysates from cells treated with phosphatase inhibitors versus phosphatase-treated samples to confirm specificity for the phosphorylated form of the protein .

How can specificity of Phospho-NFKB2 (Ser866) antibodies be validated in experimental systems?

Validating the specificity of Phospho-NFKB2 (Ser866) antibodies requires a multi-faceted approach to ensure that observed signals genuinely represent the phosphorylated epitope. Researchers should employ the following comprehensive validation strategy:

• Phosphatase Treatment Control: Treat duplicate samples with lambda phosphatase prior to immunoblotting. The phospho-specific signal should disappear in treated samples while total NF-κB2 signal (detected with a separate antibody) remains unchanged .

• Peptide Competition Assay: Pre-incubate the antibody with excess phospho-peptide containing the Ser866 sequence (E-D-S(p)-A-Y) versus non-phosphorylated peptide. Specific signal should be blocked only by the phospho-peptide .

• Mutation Studies: Compare signals between wild-type cells and those expressing S866A mutant NF-κB2, which cannot be phosphorylated at this position. The absence of signal in mutant samples confirms specificity.

• Pathway Modulation: Treat cells with NIK or IKKα inhibitors to prevent phosphorylation, or stimulate the non-canonical pathway with relevant ligands. Changes in signal intensity should correlate with expected pathway activity.

• Cross-Reactivity Assessment: Test the antibody against samples containing other phosphorylated proteins with similar flanking sequences to ensure no cross-reactivity occurs.

• Transfection Controls: Compare endogenous signals with those from cells transfected with either wild-type or phospho-mutant NF-κB2 expression constructs. Antibodies showing "Transfected Only" sensitivity should be used with appropriate caution .

Documentation of these validation steps enhances experimental rigor and supports the reliability of research findings involving phosphorylation-specific detection.

What are the key differences in protocols for using Phospho-NFKB2 (Ser866) antibodies across various application techniques?

Using Phospho-NFKB2 (Ser866) antibodies across different applications requires technique-specific protocol adjustments to optimize signal detection while maintaining specificity:

Western Blotting (WB):

• Dilution: 1:1000 for standard detection

• Blocking: 5% BSA in TBST (not milk, which contains phosphatases)

• Membrane: PVDF preferred for phospho-proteins

• Incubation: Overnight at 4°C with gentle agitation

• Detection: Enhanced chemiluminescence with extended exposure times

• Expected band: ~110 kDa for phosphorylated p100

Immunoprecipitation (IP):

• Dilution: 1:50 for capture of phospho-proteins

• Pre-clearing: Essential to reduce non-specific binding

• Protein A/G beads: Pre-block with BSA

• Lysate amount: 500-1000 μg of total protein

• Incubation: 4 hours to overnight at 4°C

• Washing: Multiple stringent washes to reduce background

Immunohistochemistry (IHC):

• Dilution: 1:50-1:100

• Fixation: 10% neutral buffered formalin preferred

• Antigen retrieval: Critical step, using citrate buffer pH 6.0

• Signal amplification: Often required due to low abundance

• Counterstaining: Light hematoxylin to visualize tissue architecture

• Controls: Adjacent sections with non-phospho antibody

Immunofluorescence (IF):

• Dilution: 1:50-1:100

• Fixation: 4% paraformaldehyde for 15 minutes

• Permeabilization: 0.2% Triton X-100 for 10 minutes

• Blocking: 2% BSA, 5% normal serum, 0.1% Tween-20

• Secondary antibody: Fluorophore-conjugated anti-rabbit IgG

• Nuclear counterstain: DAPI at 1:1000 dilution

Each application requires specific attention to phospho-epitope preservation, with temperature control, phosphatase inhibition, and validation controls being universally important across all techniques.

How can researchers address weak or inconsistent signals when using Phospho-NFKB2 (Ser866) antibodies?

Researchers encountering weak or inconsistent signals with Phospho-NFKB2 (Ser866) antibodies should implement a systematic troubleshooting approach:

• Phosphatase Activity Control: Phosphorylation at Ser866 is highly labile. Ensure complete phosphatase inhibition by:

  • Using fresh phosphatase inhibitor cocktails

  • Increasing inhibitor concentrations in lysis buffers

  • Maintaining samples at 4°C throughout processing

  • Adding additional phosphatase inhibitors (e.g., calyculin A at 100 nM)

• Stimulation Optimization: The Ser866 phosphorylation is typically stimulus-dependent. Optimize by:

  • Testing multiple stimulation timepoints (2-24 hours)

  • Adjusting concentrations of pathway activators

  • Using positive control stimuli (e.g., lymphotoxin-β receptor agonists)

  • Considering cell-specific pathway activation requirements

• Antibody Concentration Adjustment: Due to the potentially low abundance of phosphorylated p100:

  • Test multiple antibody dilutions, potentially using higher concentrations than standard (1:500 to 1:100)

  • Extend primary antibody incubation time to 24-48 hours at 4°C

  • Use more sensitive detection systems (e.g., femto-level ECL substrates)

• Sample Enrichment: For low-abundance targets:

  • Increase loading amount to 50-100 μg protein per lane

  • Perform immunoprecipitation before Western blotting

  • Use phospho-protein enrichment columns prior to analysis

• Technical Validation: Ensure proper technique by:

  • Running fresh positive controls from cells with known pathway activation

  • Testing antibody lot performance with control lysates

  • Verifying transfer efficiency with reversible staining

Implementing these approaches systematically can help resolve signal issues while maintaining experimental rigor and specificity.

What are the most critical experimental controls when investigating NFKB2 phosphorylation dynamics?

When investigating NFKB2 phosphorylation dynamics, implementing comprehensive controls is essential for experimental validity and interpretability. The following controls address the most critical aspects of phosphorylation analysis:

• Pathway Activation Controls:

  • Positive Stimulation: Cells treated with established non-canonical pathway activators (e.g., BAFF, CD40L)

  • Kinase Inhibition: Parallel samples treated with NIK or IKKα inhibitors to block phosphorylation

  • Time Course: Sequential sampling to establish phosphorylation kinetics following stimulation

• Phosphorylation-Specific Controls:

  • Phosphatase-Treated Samples: Duplicate samples treated with lambda phosphatase to demonstrate phospho-specificity

  • Phospho-Null Mutants: Cells expressing S866A mutant NF-κB2 as negative controls

  • Total Protein Detection: Parallel blots with antibodies against total NF-κB2 to normalize phospho-signals

• Processing Controls:

  • Proteasome Inhibition: MG132-treated samples to accumulate phosphorylated intermediates

  • Ubiquitination Assessment: Co-immunoprecipitation to detect ubiquitinated p100 following phosphorylation

  • p100/p52 Ratio Analysis: Simultaneous detection of precursor and processed forms to assess conversion efficiency

• Cellular Context Controls:

  • Cell Type Comparison: Parallel analysis in multiple relevant cell types (e.g., B cells, T cells, fibroblasts)

  • Patient-Derived Materials: Where available, samples from patients with known NFKB2 mutations

  • Wild-Type/Knockout Comparison: Samples from wild-type versus NFKB2-modified animals

• Technical Controls:

  • Antibody Specificity: Peptide competition controls with phospho and non-phospho peptides

  • Loading Controls: Multiple housekeeping proteins to ensure equal loading

  • Reproducibility Assessment: Biological replicates across independent experiments

How can researchers quantitatively assess the ratio of phosphorylated to total NFKB2 protein?

Quantitative assessment of the phosphorylated to total NFKB2 protein ratio requires precise methodological approaches to generate reliable metrics. Researchers should implement the following strategies:

• Western Blot-Based Quantification:

  • Run duplicate blots or strip and reprobe membranes with antibodies against phospho-Ser866 and total NF-κB2

  • Use fluorescent secondary antibodies with different wavelengths for simultaneous detection

  • Employ densitometry software (ImageJ, Image Studio, etc.) to measure band intensities

  • Calculate the ratio of phospho-Ser866 signal to total NF-κB2 signal for each sample

  • Include a standard curve of recombinant phosphorylated and non-phosphorylated protein for absolute quantification

• ELISA-Based Approaches:

  • Develop sandwich ELISAs using a capture antibody against total NF-κB2 and detection with either phospho-specific or another total protein antibody

  • Calculate the ratio of signal from phospho-detection to total protein detection

  • Normalize against standard curves of known quantities

• Flow Cytometry:

  • Perform intracellular staining with fluorophore-conjugated antibodies against both phospho-Ser866 and total NF-κB2

  • Analyze the mean fluorescence intensity ratio on a per-cell basis

  • Sort cell populations based on phosphorylation status for further analysis

• Mass Spectrometry:

  • Employ quantitative phosphoproteomics using SILAC or TMT labeling

  • Isolate NF-κB2 by immunoprecipitation before analysis

  • Calculate the stoichiometry of phosphorylation at Ser866 compared to the unmodified peptide

  • Perform targeted SRM/MRM assays for high sensitivity detection of specific phosphopeptides

• Image-Based Analysis:

  • Perform immunofluorescence with phospho-Ser866 and total NF-κB2 antibodies

  • Acquire images using consistent exposure settings

  • Analyze colocalization and intensity ratios at the single-cell level

  • Measure nuclear versus cytoplasmic distribution to assess functional consequences of phosphorylation

For any quantitative approach, researchers should:

• Include appropriate statistical analysis (minimum of 3 biological replicates)

• Report both raw values and normalized ratios

• Establish thresholds for defining "high" versus "low" phosphorylation states based on biological contexts

These methodologies enable researchers to move beyond qualitative assessments to precise quantitative measurements of phosphorylation stoichiometry, facilitating more nuanced understanding of pathway dynamics.

How do mutations near the Ser866 phosphorylation site contribute to human immune disorders?

Mutations near the Ser866 phosphorylation site of NFKB2 have emerged as significant contributors to human immune disorders through disruption of the tightly regulated non-canonical NF-κB pathway. These mutations typically affect the processing of p100 to p52, leading to imbalanced signaling with profound immunological consequences.

Clinical research has identified patients with heterozygous mutations that truncate the NF-κB2 precursor protein proximal to the degron region, which includes the Ser866 phosphorylation site . These mutations enable signal-independent production of p52, resulting in various immunodeficiencies and cytopenias . A particularly informative case involved a patient with a de novo T-nucleotide insertion at c.2598 of NFKB2, creating a frameshift mutation (p.A867Cfs*) adjacent to the Ser866 phosphorylation site . This mutation was associated with combined immune deficiency, demonstrating that aberrant NFKB2 signaling affects both humoral immunity and TCR-mediated T cell proliferation .

Mouse models carrying mutations in this region further illuminate the mechanistic basis of these disorders. The S866fs NFKB2 variant, which causes a net deletion of one residue between Ser866 and Ser870, leads to altered p100/p52 ratios . This suggests that precise phosphorylation at Ser866 and nearby residues maintains a critical threshold of p100 degradation that, when disrupted, leads to immune dysregulation.

The emerging understanding suggests a delicate balance where:

• Complete loss of processing (as in Lym1 mutants) leads to severe immune deficiency

• Partial disruption of processing (as in S866fs variants) results in intermediate phenotypes

• Hyperactive processing can lead to inflammatory conditions

These findings highlight the potential for developing targeted therapies based on precise understanding of phosphorylation-dependent p100 processing thresholds in various immune disorders.

What are the latest techniques for simultaneous detection of multiple phosphorylation events in the NFKB2 regulatory domain?

Recent technological advances have significantly enhanced capabilities for simultaneous detection of multiple phosphorylation events in the NFKB2 regulatory domain, offering unprecedented insights into signaling dynamics. Researchers now employ several cutting-edge approaches:

• Multiplex Phospho-Flow Cytometry:

  • Utilizes antibodies against different phosphorylation sites (Ser866, Ser870, Ser872) conjugated to distinct fluorophores

  • Enables single-cell analysis of phosphorylation patterns

  • Allows correlation of multiple phosphorylation events with cellular phenotypes

  • Provides statistical power through high-throughput analysis of thousands of cells

• Mass Spectrometry-Based Phosphoproteomics:

  • Targeted parallel reaction monitoring (PRM) for specific phosphopeptides

  • Analysis of phosphorylation site stoichiometry and interdependence

  • Identification of novel phosphorylation sites within the regulatory domain

  • Temporal dynamics of multiple phosphorylation events following stimulus

• Proximity Ligation Assays (PLA):

  • Detection of spatial relationships between phosphorylation events

  • Visualization of phosphorylation patterns in fixed cells and tissues

  • Assessment of co-occurrence of multiple modifications on individual protein molecules

  • Combination with super-resolution microscopy for nanoscale mapping

• Engineered FRET-Based Biosensors:

  • Genetically encoded reporters sensitive to specific phosphorylation events

  • Real-time visualization of phosphorylation dynamics in living cells

  • Measurement of phosphorylation kinetics with subcellular resolution

  • Correlation of phosphorylation with protein conformation changes

• Nanobody-Based Detection Systems:

  • Highly specific recognition of individual phosphorylation states

  • Multiplexed detection with minimal steric hindrance

  • Compatible with intracellular expression for live-cell studies

  • Potential for super-resolution phosphorylation mapping

These advanced methodologies have revealed that phosphorylation at Ser866 often occurs in coordinated patterns with modifications at Ser870 and other sites, suggesting a "phosphorylation code" that determines the fate and function of NF-κB2. Such integrated approaches are essential for understanding the complex regulatory mechanisms governing the non-canonical NF-κB pathway in both normal physiology and disease states.

How does crosstalk between canonical and non-canonical NF-κB pathways influence Ser866 phosphorylation status?

The intricate crosstalk between canonical and non-canonical NF-κB pathways creates a complex regulatory network that significantly influences Ser866 phosphorylation status of NF-κB2/p100. This interplay operates through multiple mechanistic layers:

• Shared Upstream Regulators:

  • Certain stimuli (e.g., TNF-α, LPS) can activate both pathways with different kinetics

  • Canonical pathway activation can induce expression of non-canonical pathway components

  • NIK, primarily associated with non-canonical signaling, can contribute to canonical pathway in specific contexts

• Regulatory Protein Competition:

  • IKKα, required for Ser866 phosphorylation, participates in both pathways

  • During strong canonical activation, sequestration of IKKα in the canonical IKK complex may limit availability for p100 phosphorylation

  • p100 itself acts as an IκB protein, potentially inhibiting canonical pathway components

• Feedback Mechanisms:

  • Canonical pathway activation induces expression of p100, providing substrate for subsequent non-canonical processing

  • Processed p52 can regulate transcription of genes involved in canonical pathway regulation

  • Both pathways induce negative regulators that can affect the alternative pathway

• Phosphorylation Dynamics:

  • Canonical pathway components (IKKβ, IKKγ) may contribute to phosphorylation at sites near Ser866 under specific conditions

  • Ser866 phosphorylation status can be affected by prior phosphorylation events mediated by canonical pathway kinases

  • Phosphatases regulated by canonical signaling may modulate Ser866 phosphorylation persistence

• Pathological Relevance:

  • In chronic inflammatory conditions, persistent canonical pathway activation alters the threshold for Ser866 phosphorylation

  • Cancer-associated mutations in canonical pathway components indirectly affect non-canonical processing through altered crosstalk

  • Therapeutic targeting of one pathway inevitably affects the other through these interconnected mechanisms

The dynamic equilibrium between these pathways creates context-specific patterns of Ser866 phosphorylation that cannot be understood by studying the non-canonical pathway in isolation. This integrated understanding has significant implications for developing pathway-specific therapeutics and interpreting experimental results involving NF-κB signaling.