Phospho-NFKB2 (Ser870) Antibody

What is the biological significance of NFKB2/p100 Ser870 phosphorylation?

Ser870 represents one of several critical phosphorylation sites in the p100 degron region that regulates noncanonical NF-κB signaling. During pathway activation, NF-κB–inducing kinase (NIK) cooperates with IKKα to phosphorylate the p100 degron at specific sites (Ser866, Ser870, and Ser872) before ubiquitination of Lys855, which tags the C-terminal portion of p100 for proteasomal removal . This phosphorylation is essential for the partial processing of p100 to p52, enabling proper noncanonical NF-κB signaling.

How does Ser870 phosphorylation differ functionally from other nearby phosphorylation sites like Ser872?

While Ser870 and Ser872 are both located within the degron region of p100, they appear to have complementary roles in regulating p100 processing. The proximity of these sites suggests they work cooperatively to facilitate proper protein degradation. Research indicates that mutations affecting this region can lead to accumulation of p100 and disrupted p100/p52 ratios . For comprehensive pathway analysis, researchers often need to examine multiple phosphorylation sites to understand their collective and individual contributions to signaling outcomes.

What are the key specifications to consider when selecting a Phospho-NFKB2 (Ser870) antibody?

When selecting a Phospho-NFKB2 (Ser870) antibody, researchers should consider:

Cross-referencing these specifications with your experimental design is essential for obtaining reliable results.

How do affinity-purified phospho-specific antibodies enhance experimental outcomes?

Affinity-purified phospho-specific antibodies, like those targeting Phospho-NFKB2 (Ser870), undergo a two-step purification process that significantly enhances their specificity. These antibodies are typically purified on phosphopeptide columns, with non-phosphopeptide-reactive antibodies removed by chromatography using non-phosphorylated peptides . This process yields antibodies that recognize only the phosphorylated form of the protein, minimizing background and cross-reactivity with unphosphorylated epitopes. This enhanced specificity is critical for accurately differentiating between phosphorylated and non-phosphorylated states in various applications.

What are the optimal protocols for detecting Phospho-NFKB2 (Ser870) in Western blot applications?

For optimal Western blot detection of Phospho-NFKB2 (Ser870):

• Sample preparation:

  • Include phosphatase inhibitors in lysis buffers to preserve phosphorylation

  • Maintain samples at 4°C throughout processing

  • Consider using positive controls like RAW264.7 cells treated with EGF (200ng/ml for 30 minutes)

• Recommended protocol:

  • Primary antibody dilution: 1:500-1:2000 (optimize for your specific antibody)

  • Incubation: 4°C overnight for primary antibody

  • Secondary antibody dilution: Approximately 1:10000

  • Secondary antibody incubation: 37°C for 1 hour

  • Expected molecular weight: ~97 kDa (for full-length phosphorylated p100)

• Controls:

  • Include both phosphorylated (stimulated) and non-phosphorylated (unstimulated) samples

  • Consider using phosphatase treatment of duplicate samples as negative controls

How can immunohistochemistry (IHC) with Phospho-NFKB2 (Ser870) antibodies provide insights into tissue-specific activation patterns?

IHC using Phospho-NFKB2 (Ser870) antibodies enables visualization of noncanonical NF-κB activation patterns across different tissues and cell types. This approach allows researchers to:

• Identify cell-specific activation within heterogeneous tissues

• Track spatial distribution of noncanonical NF-κB signaling in disease models

• Correlate phosphorylation patterns with histopathological features

For optimal IHC results:

• Test multiple antigen retrieval methods (heat-induced vs. enzymatic)

• Optimize antibody concentration through dilution series

• Include positive control tissues (lymphoid tissues often show detectable levels)

• Perform parallel staining with total NFKB2 antibodies to normalize phospho-signals

This approach has been particularly valuable in examining thymic medullary development abnormalities associated with NFKB2 mutations .

How should researchers interpret changes in the p100/p52 ratio when studying Phospho-NFKB2 (Ser870)?

• Mutations affecting phosphorylation sites in the degron region (including Ser870) typically increase the p100/p52 ratio by inhibiting processing

• The p100/p52 ratio correlates negatively with lifespan in mouse models with NFKB2 mutations

• Homozygous mutations generally cause more dramatic increases in this ratio than heterozygous mutations

When analyzing experimental data, researchers should compare p100/p52 ratios under both basal and stimulated conditions to assess pathway responsiveness.

What controls are essential when using Phospho-NFKB2 (Ser870) antibodies?

To ensure data reliability when using Phospho-NFKB2 (Ser870) antibodies, the following controls are essential:

• Positive controls:

  • Cell lines treated with known activators of noncanonical NF-κB signaling (e.g., RAW264.7 cells treated with EGF)

  • Tissues from conditions known to activate noncanonical NF-κB signaling

• Negative controls:

  • Lambda phosphatase-treated duplicate samples to confirm phospho-specificity

  • Samples from knockout models (when available) or siRNA-treated cells

  • Blocking with immunizing phosphopeptide to confirm antibody specificity

• Technical controls:

  • Total NFKB2/p100 antibody staining on parallel samples to normalize phospho-signals

  • Multiple antibody dilutions to ensure detection is in the linear range

  • Consistent loading controls across all samples

These controls collectively ensure that observed signals are specific to phosphorylated Ser870 rather than artifacts or non-specific binding.

How can Phospho-NFKB2 (Ser870) antibodies be used to study the relationship between noncanonical NF-κB signaling and autoimmunity?

Phospho-NFKB2 (Ser870) antibodies provide valuable tools for investigating the link between noncanonical NF-κB signaling disruptions and autoimmune pathologies. Research has demonstrated that mutations affecting the p100 degron region (which includes Ser870) can lead to:

• Thymic medullary hypoplasia and disrupted T-cell selection

• Altered balance of strongly TCR-signaled Helios+CCR7- cells and weakly TCR-signaled Helios-CCR7+ cells

• TCR repertoires enriched in hydrophobic motifs, a biomarker of self-reactivity

• Multiorgan autoimmunity in homozygous mutant mice

Methodological approaches for studying these connections include:

• Temporal analysis of phosphorylation patterns during disease progression

• Correlation of phosphorylation levels with autoantibody production

• Examination of phosphorylation in specific immune cell subsets

• Therapeutic interventions targeting this pathway followed by phosphorylation assessment

What insights can be gained by comparing different phosphorylation sites (Ser866, Ser870, Ser872) in the p100 degron region?

Comparing phosphorylation patterns across the p100 degron region can provide crucial insights into signal integration and processing efficiency:

This comparative approach can reveal how cells integrate multiple signals to fine-tune noncanonical NF-κB activity in different physiological contexts.

How can researchers address weak or inconsistent Phospho-NFKB2 (Ser870) signals in Western blot applications?

When encountering weak or inconsistent signals:

• Sample preparation optimization:

  • Ensure complete protease and phosphatase inhibition during lysis

  • Optimize cell stimulation protocols (timing, concentration of stimulants)

  • Consider subcellular fractionation to enrich for nuclear components

• Technical adjustments:

  • Increase protein loading (up to 50-75μg) if signals are weak

  • Extend primary antibody incubation time (up to 48 hours at 4°C)

  • Try alternative transfer methods (wet transfer vs. semi-dry)

  • Use high-sensitivity ECL substrate for detection

• Antibody optimization:

  • Test lower dilutions of primary antibody (e.g., 1:500)

  • Extend wash steps to reduce background

  • Consider alternative secondary antibodies with higher sensitivity

• Biological considerations:

  • Verify pathway activation using additional markers of noncanonical NF-κB signaling

  • Consider cell type-specific differences in signaling kinetics and magnitude

What methodological precautions are necessary when studying phosphorylation dynamics of NFKB2/p100?

When investigating phosphorylation dynamics:

• Temporal considerations:

  • Design detailed time-course experiments (e.g., 0, 15, 30, 60, 120, 240 minutes post-stimulation)

  • Use rapid cell harvesting techniques to capture transient phosphorylation events

  • Consider "pulse-chase" approaches to track the fate of phosphorylated proteins

• Sample handling:

  • Maintain samples at 4°C throughout processing

  • Include both general and site-specific phosphatase inhibitors in all buffers

  • Process samples immediately after collection

• Controls for pathway specificity:

  • Include pathway inhibitors (e.g., NIK or IKKα inhibitors) to confirm specificity

  • Use cells with genetic knockouts of upstream pathway components

  • Consider the effects of general stressors that might indirectly affect phosphorylation

• Quantification approaches:

  • Always normalize phospho-signals to total protein levels

  • Use appropriate loading controls

  • Employ multiple technical and biological replicates to account for variability

These precautions are essential for accurately capturing the dynamic nature of NFKB2/p100 phosphorylation events, which can be both rapid and transient.