Phospho-IKBKG (S85) Antibody

What is IKBKG and what is the significance of S85 phosphorylation?

IKBKG (also known as NEMO, NF-kappa-B essential modulator) functions as the regulatory subunit of the IKK core complex. This complex phosphorylates inhibitors of NF-kappa-B, leading to their dissociation and subsequent degradation, ultimately activating the NF-κB pathway . The phosphorylation of IKBKG at serine 85 (S85) represents a critical post-translational modification that regulates its activity.

IKBKG's binding to scaffolding polyubiquitin plays a key role in IKK activation across multiple signaling receptor pathways . It can recognize and bind both 'Lys-63'-linked and linear polyubiquitin upon cell stimulation, with higher affinity for linear polyubiquitin . Beyond NF-κB activation, IKBKG is implicated in viral activation of IRF3 and TLR3-mediated antiviral innate responses .

S85 phosphorylation specifically modulates IKBKG's role in signal transduction, with phosphorylation increasing rapidly following TNFα stimulation, as demonstrated in HEK293T cells treated with TNFα for various time intervals .

What are the primary research applications for Phospho-IKBKG (S85) Antibody?

Phospho-IKBKG (S85) antibodies are instrumental in multiple research applications focusing on NF-κB signaling. Primary applications include:

• Investigation of NF-κB pathway activation dynamics: These antibodies enable temporal monitoring of IKBKG phosphorylation following stimulation with cytokines, particularly TNFα, allowing researchers to track signaling pathway activation kinetics .

• Analysis of immune response mechanisms: Given IKBKG's critical role in immune signaling, these antibodies help evaluate how various immune stimuli affect NF-κB activation through IKBKG phosphorylation .

• Characterization of disease-associated mutations: Phospho-specific antibodies can help determine how mutations in IKBKG affect its phosphorylation state and subsequent signaling capacity, particularly in immunodeficiency contexts .

• Drug effect studies: These antibodies are valuable tools for examining how tyrosine kinase inhibitors (TKIs) and other therapeutic compounds impact IKBKG phosphorylation and downstream signaling events .

When designing experiments, researchers should include appropriate positive controls (e.g., TNFα-stimulated cell lysates) and negative controls (e.g., unstimulated lysates or phosphatase-treated samples) to validate antibody specificity .

What experimental techniques are compatible with Phospho-IKBKG (S85) Antibody?

Based on the available research data, Phospho-IKBKG (S85) antibody demonstrates compatibility with several experimental techniques:

• Western Blot (WB): The primary application for detection of phosphorylated IKBKG. Recommended dilution ranges from 1:500 to 1:3000 depending on the specific antibody and experimental conditions .

• Immunohistochemistry (IHC): Some phospho-IKBKG antibodies can be used for tissue section analysis with recommended dilutions between 1:20 and 1:200 .

• Immunofluorescence (IF/ICC): For cellular localization studies, with typical dilutions of 1:50 to 1:500 .

• Co-Immunoprecipitation (Co-IP): Used to study protein-protein interactions involving phosphorylated IKBKG .

• ELISA: For quantitative assessment of phosphorylated IKBKG levels .

The experimental compatibility varies between different commercial antibodies, and researchers should validate each application for their specific research context.

How do you optimize Western blot protocols for detecting Phospho-IKBKG (S85)?

Optimizing Western blot protocols for Phospho-IKBKG (S85) detection requires careful attention to several key parameters:

• Sample preparation:

  • Immediately add phosphatase inhibitors to lysis buffers to prevent dephosphorylation

  • Use fresh samples when possible, as freeze-thaw cycles can affect phosphorylation status

  • Include both positive controls (TNFα-stimulated cells) and negative controls in experimental design

• Gel electrophoresis and transfer:

  • Use freshly prepared buffers

  • Ensure complete protein transfer, particularly for higher molecular weight proteins

  • Consider using PVDF membranes which may provide better signal for phospho-epitopes

• Antibody incubation:

  • Start with manufacturer's recommended dilution (typically 1:500 to 1:1000 for Phospho-IKBKG S85)

  • Optimize incubation temperature and duration (typically 4°C overnight yields better results)

  • Use 5% BSA in TBST as blocking solution rather than milk, as milk contains phospho-proteins and phosphatases

• Signal detection:

  • Consider using enhanced chemiluminescence (ECL) substrates specifically designed for phospho-protein detection

  • Include loading controls and total IKBKG detection on separate blots or after stripping

This optimization approach has been validated with various cell types including HEK293T, A549, and mouse and rat tissue lysates .

What are the essential controls and validation approaches for Phospho-IKBKG (S85) Antibody experiments?

Rigorous validation is critical for phospho-specific antibody experiments. Essential controls include:

• Positive controls:

  • TNFα-stimulated cell lysates (20ng/ml TNFα for varying time points: 5 minutes, 20 minutes, 40 minutes, and 4 hours)

  • Lysates from cells with known IKBKG pathway activation

• Negative controls:

  • Untreated/unstimulated cells

  • Phosphatase-treated lysates to remove phosphorylation

  • IKBKG-deficient cell lines (particularly important for determining antibody specificity)

• Validation approaches:

  • Peptide competition assays using phosphorylated and non-phosphorylated peptides

  • Antibody validation in IKBKG knock-out/knock-down models

  • Correlation with other readouts of NF-κB pathway activation (e.g., p65 nuclear translocation, IκBα degradation)

  • Comparison of results across multiple detection methods (Western blot, immunofluorescence)

• Experimental validation:

  • Dose-response and time-course analyses to establish phosphorylation dynamics

  • Testing across multiple cell types to confirm antibody performance in different experimental contexts

Data from these validation experiments should be systematically documented to ensure reproducibility and reliability of results.

How does IKBKG S85 phosphorylation relate to NF-κB pathway activation and other signaling events?

IKBKG S85 phosphorylation is intricately connected to NF-κB pathway activation through several mechanisms:

• Position in the signaling cascade:

  • IKBKG functions as the regulatory subunit of the IKK complex, which is central to NF-κB activation

  • S85 phosphorylation modulates IKBKG's activity within this complex

  • This phosphorylation can occur in response to various stimuli, particularly TNFα

• Relationship to IκBα degradation:

  • While IκBα degradation is considered a primary requirement for NEMO-mediated immune signaling, research shows that some IKBKG mutations can impair immune signaling without affecting IκBα degradation

  • In the panr2 mouse model, despite normal IκBα degradation, ERK phosphorylation and NF-κB p65 nuclear translocation were severely impaired

  • This suggests that IKBKG-regulated pathways extend beyond IκBα degradation

• Cross-talk with other pathways:

  • IKBKG phosphorylation affects both NF-κB and MAPK signaling

  • Phosphorylation of p105, MEK, and ERK can be impaired when IKBKG function is compromised

  • IKBKG participates in crosstalk between EGF/EGFR signaling and other pathways like glycolysis and transmembrane transport

• Integration of multiple signals:

  • IKBKG can be regulated by different upstream kinases depending on the stimulus

  • The phosphorylation state of IKBKG reflects integration of multiple input signals

Understanding these relationships is essential for interpreting experimental results involving Phospho-IKBKG (S85) detection.

How can researchers study the temporal dynamics of IKBKG S85 phosphorylation in response to different stimuli?

Investigating temporal dynamics of IKBKG S85 phosphorylation requires sophisticated experimental approaches:

• Time-course stimulation experiments:

  • Treat cells with stimuli (e.g., TNFα, LPS, IL-1β) for precisely timed intervals ranging from minutes to hours

  • Example: TNFα stimulation at 5 minutes, 20 minutes, 40 minutes, and 4 hours has shown differential IKBKG S85 phosphorylation patterns

  • Synchronize cell populations prior to stimulation for more uniform responses

• Quantitative Western blot analysis:

  • Use digital imaging systems that provide linear detection ranges

  • Normalize phospho-signal to total IKBKG protein

  • Generate quantitative curves showing phosphorylation kinetics

• Live-cell imaging techniques:

  • Develop FRET-based biosensors for IKBKG phosphorylation

  • Use phospho-specific antibodies conjugated to fluorescent tags for immunofluorescence time-course studies

  • Combine with subcellular fractionation to track IKBKG localization in relation to phosphorylation

• Single-cell analysis:

  • Flow cytometry or mass cytometry using phospho-specific antibodies

  • Single-cell Western techniques for heterogeneous cell populations

  • Correlation with other signaling events at single-cell resolution

• Mathematical modeling:

  • Develop computational models integrating experimental data on phosphorylation dynamics

  • Use differential equations to describe phosphorylation/dephosphorylation kinetics

  • Predict responses to novel stimuli or combination treatments

These approaches have revealed that IKBKG phosphorylation dynamics depend on stimulus type, duration, and cellular context, with rapid responses to TNFα and more complex patterns in response to other immune activators.

How do mutations in IKBKG affect S85 phosphorylation and what are the implications for immune signaling?

Mutations in IKBKG have profound effects on its phosphorylation and subsequent immune signaling capabilities:

• Types of mutations and their effects:

  • Loss-of-function mutations (e.g., p.Tyr308*) can completely inhibit NF-κB activity even when stimulated with lipopolysaccharide

  • Hypomorphic alleles typically cause syndromes of immune deficiency and ectodermal dysplasia

  • Some mutations selectively impair specific IKBKG functions without affecting others

• Differential impact on signaling pathways:

  • Certain mutations can impair Toll-like receptor signaling, lymph node formation, and development of memory and regulatory T cells

  • Some mutations allow normal IκBα degradation yet impair ERK phosphorylation and NF-κB p65 nuclear translocation

  • This selective impairment highlights the importance of NEMO-regulated pathways beyond IκBα degradation

• Methodological approaches to study mutation effects:

  • Long-range PCR and Sanger sequencing for detecting IKBKG mutations (complex due to pseudogene presence)

  • Dual-luciferase reporter assays to assess NF-κB activity with mutant IKBKG proteins

  • Immunoblot analysis to measure phosphorylation of downstream targets like p65 and degradation of IκBα

  • Lipopolysaccharide stimulation assays to evaluate functional responses

• Clinical correlations:

  • Mosaic variants with less than 30% mosaicism can cause immune dysregulation

  • Gain-of-function variants can lead to increased phosphorylation of NF-κB and enhanced production of pro-inflammatory cytokines

  • Patient-derived iPSCs differentiated into myeloid cells show increased NF-κB phosphorylation in response to TLR stimulation

These findings demonstrate how crucial proper IKBKG phosphorylation is for immune function and how mutations that affect phosphorylation can lead to immunodeficiency and other disorders.

What methods can be used to study the interplay between IKBKG S85 phosphorylation and other post-translational modifications?

Understanding the complex interplay between different post-translational modifications (PTMs) on IKBKG requires integrative methodologies:

• Sequential enrichment of post-translational modifications (SEPTM) proteomics:

  • This technique can identify multiple PTMs including phosphorylation, ubiquitination, and acetylation on the same protein

  • Has been successfully used to identify 12,461 unique PTMs in response to tyrosine kinase inhibitors

  • Can reveal how different PTMs are affected by various treatments

• Mass spectrometry-based approaches:

  • Phospho-enrichment followed by ubiquitin enrichment (or vice versa)

  • Multi-dimensional fractionation to separate peptides with different modifications

  • Label-free quantification or SILAC/TMT labeling for quantitative comparisons

  • Analysis of PTM stoichiometry and site occupancy

• Site-directed mutagenesis studies:

  • Generate IKBKG mutants where specific modification sites are altered (e.g., S85A, K399R)

  • Study how modification at one site affects modifications at other sites

  • Evaluate functional consequences of preventing specific modifications

• Time-resolved studies of multiple PTMs:

  • Monitor phosphorylation, ubiquitination, and acetylation dynamics in parallel

  • Establish temporal relationships between different modifications

  • Research shows phosphorylation and ubiquitination often display asymmetric responses to different treatments

• Co-immunoprecipitation with different modification-specific antibodies:

  • Sequential IPs to isolate proteins with multiple specific modifications

  • Analysis of protein complexes formed with differently modified IKBKG variants

• Computational network modeling:

  • Construction of Co-Cluster Correlation Networks (CCCN) and Cluster Filtered Networks (CFN) from PTM data

  • Analysis of pathway crosstalk and "mutual friends" between signaling pathways

  • Integration of multiple PTM datasets to identify common network features

These advanced approaches have revealed that different PTMs on IKBKG respond asymmetrically to different treatments, suggesting complex regulatory mechanisms governing IKBKG function in various contexts.

How can phospho-proteomic approaches be used to understand IKBKG S85 phosphorylation in broader cellular signaling networks?

Integrating IKBKG S85 phosphorylation data into broader signaling networks requires sophisticated phospho-proteomic and computational approaches:

• Global phospho-proteomic profiling:

  • Titanium dioxide (TiO₂) or immobilized metal affinity chromatography (IMAC) for phosphopeptide enrichment

  • Tandem mass spectrometry (MS/MS) for identification and quantification

  • IKBKG phosphorylation can be contextualized within thousands of other phosphorylation events

• Network construction methodologies:

  • Co-Cluster Correlation Network (CCCN) construction using dimension reduction algorithms like t-distributed stochastic neighbor embedding (t-SNE)

  • Cluster Filtered Network (CFN) development to identify functional relationships

  • Analysis of composite shortest paths connecting different pathways, such as between EGF/EGFR signaling and other cellular processes

• Pathway crosstalk analysis:

  • Identification of "mutual friends" – nodes connecting to members of different pathways

  • Analysis of direct connections between pathways (e.g., nine proteins in EGF/EGFR signaling pathway interact directly with eight proteins in transmembrane transport pathway)

  • Focus on connections where both interaction partners show PTM changes in response to stimuli

• Integration with other 'omics data:

  • Correlation of phosphorylation changes with transcriptomic alterations

  • Integration with ubiquitylome and acetylome data

  • Development of multi-omics visualizations and analyses

• Functional validation of network predictions:

  • Targeted inhibition or activation of nodes predicted to affect IKBKG phosphorylation

  • Measurement of signaling dynamics after perturbation of network components

  • Evaluation of cellular outcomes in relation to network changes

Research using these approaches has revealed that IKBKG phosphorylation at S85 is integrated within complex cellular networks, with connections to diverse processes including glycolysis, transmembrane transport, and immune signaling . These networks provide a systems-level understanding of how IKBKG phosphorylation contributes to cellular function and disease.