Phospho-IKBKG (S376) Antibody

What is IKBKG and what is its role in cellular signaling?

IKBKG (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma), also known as NEMO (NF-kappa-B essential modifier), functions as the regulatory subunit of the IKK complex that activates NF-kappaB. It plays a critical role in the NF-κB signaling pathway, which regulates genes involved in inflammation, immunity, and cell survival . IKBKG has several aliases including FIP-3, IkB kinase-associated protein 1 (IKKAP1), and IKK-gamma . As part of the NF-κB regulatory complex or signalsome, IKBKG works with closely related serine/threonine kinases IKKα and IKKβ to phosphorylate IκB proteins, targeting them for degradation and thus releasing NF-κB to translocate to the nucleus .

Why is the phosphorylation at Serine 376 of IKBKG significant?

Phosphorylation at Serine 376 represents a specific post-translational modification of IKBKG that is involved in regulating its activity within the NF-κB signaling pathway. Research has shown that this specific phosphorylation event can be induced by stimulation with TNF-α, indicating its role in cytokine-mediated cellular responses . The S376 phosphorylation site appears to be functionally important, as evidenced by the development of site-specific antibodies and their application in studying NF-κB activation in response to various stimuli . Understanding the dynamics of this specific phosphorylation can provide insights into how inflammatory and immune signaling is regulated at the molecular level.

How does IKBKG phosphorylation relate to NF-κB pathway activation?

IKBKG phosphorylation is integrally linked to NF-κB activation. In the canonical NF-κB pathway, cytokine stimulation (such as by TNF-α) leads to IKK complex activation, which includes IKBKG phosphorylation. Active IKK complex then phosphorylates IκB, leading to its degradation and subsequent release of NF-κB for nuclear translocation . The phosphorylation at S376 of IKBKG appears to be a regulatory event in this cascade, as demonstrated by Western blot analyses showing increased phospho-IKBKG (S376) levels in cells treated with TNF-α . This phosphorylation may alter IKBKG's interaction with other components of the IKK complex or affect its conformational state, thereby modulating NF-κB signaling intensity and duration.

What experimental applications are appropriate for Phospho-IKBKG (S376) antibodies?

Phospho-IKBKG (S376) antibodies have been validated for several experimental applications:

These applications allow researchers to investigate IKBKG phosphorylation status in various experimental contexts, from protein expression levels to spatial localization within cells and tissues .

What are the optimal sample preparation methods for detecting phospho-IKBKG (S376)?

For optimal detection of phospho-IKBKG (S376), samples must be prepared with careful attention to preserving phosphorylation status:

• Cell Lysis: Use phosphatase inhibitor-containing buffers to prevent dephosphorylation. Include sodium fluoride, sodium orthovanadate, and phosphatase inhibitor cocktails in lysis buffers .

• Tissue Samples: For IHC applications, tissues should be fixed quickly after collection. Paraformaldehyde fixation followed by paraffin embedding is most common. Antigen retrieval using citrate buffer (pH 6.0) is typically required to expose the phospho-epitope .

• Protein Denaturation: For Western blotting, samples should be denatured in the presence of SDS and reducing agents at temperatures that preserve phosphorylation (70°C for 10 minutes rather than boiling) .

• Stimulation Controls: Include positive controls where cells are treated with TNF-α (20 ng/mL for 30 minutes) after overnight serum starvation to induce IKBKG phosphorylation .

• Blocking: Use BSA rather than milk for blocking Western blots, as milk contains phosphatases that may reduce signal .

Adherence to these methodological details will significantly improve the detection sensitivity and specificity of phospho-IKBKG (S376).

How can I validate the specificity of phospho-IKBKG (S376) antibody signals?

Validating antibody specificity is crucial for phospho-specific antibodies. For phospho-IKBKG (S376) antibodies, implement these validation strategies:

• Phosphatase Treatment Control: Treat duplicate samples with lambda phosphatase to remove phosphorylation and confirm signal loss.

• Stimulation-Inhibition Experiments: Compare samples from cells treated with TNF-α (which increases S376 phosphorylation) versus untreated controls . Additionally, use specific inhibitors of the NF-κB pathway to demonstrate reduced phosphorylation.

• Peptide Competition: Pre-incubate the antibody with the phosphorylated peptide immunogen to block specific binding and confirm signal reduction.

• Knockdown/Knockout Controls: Use IKBKG-depleted cells through siRNA or CRISPR methods as negative controls.

• Molecular Weight Verification: Confirm that the detected band appears at the expected molecular weight (48-52 kDa for IKBKG, though the observed weight may be 65 kDa due to post-translational modifications) .

• Examine Multiple Cell Lines: Test antibody performance across different cell lines known to express IKBKG, such as HeLa, NIH/3T3, and C6 cells .

A combination of these approaches provides robust validation of phospho-specific antibody signals.

How should I design experiments to study the dynamics of IKBKG S376 phosphorylation?

Designing experiments to study IKBKG S376 phosphorylation dynamics requires careful consideration of temporal and contextual factors:

• Time-Course Analysis: Implement staggered time points after stimulus application (e.g., TNF-α, IL-1β) to capture the onset, peak, and resolution of phosphorylation. Typical intervals include 0, 5, 15, 30, 60, and 120 minutes post-stimulation .

• Dose-Response Relationship: Test multiple concentrations of stimuli (e.g., TNF-α at 5, 10, 20, and 50 ng/mL) to determine threshold and saturation effects on S376 phosphorylation.

• Pathway Crosstalk: Examine how different upstream signaling inputs (TLR activation, cytokines, stress conditions) influence S376 phosphorylation to map pathway convergence.

• Cell Type Considerations: Compare S376 phosphorylation responses across different cell types (immune cells, epithelial cells, cancer cell lines) to identify cell-specific regulation patterns.

• Inhibitor Studies: Apply specific inhibitors at various pathway points to determine which kinases are responsible for S376 phosphorylation.

• Correlation with Functional Outputs: Pair phosphorylation measurements with functional readouts like NF-κB nuclear translocation, target gene expression, or cell survival to establish causative relationships.

Include appropriate controls in each experiment, such as serum-starved baseline conditions and positive controls (TNF-α treatment) to ensure interpretable results .

What controls should be included when using phospho-IKBKG (S376) antibodies in multiparameter analyses?

For robust multiparameter analyses with phospho-IKBKG (S376) antibodies, incorporate these essential controls:

• Phosphorylation-State Controls:

  • Positive control: TNF-α treated samples (20 ng/mL for 30 minutes)

  • Negative control: Untreated or vehicle-treated samples

  • Phosphatase-treated control: Samples treated with lambda phosphatase

• Antibody Controls:

  • Secondary-only control: Samples incubated with secondary antibody alone

  • Isotype control: Samples probed with non-specific IgG of the same host species

  • Total IKBKG antibody: Parallel detection of total IKBKG to normalize phospho-signal

• Specificity Controls:

  • Blocking peptide control: Antibody pre-incubated with phosphorylated peptide immunogen

  • Non-phosphorylated peptide control: Pre-incubation with non-phosphorylated peptide

• Technical Controls:

  • Loading control: Detection of housekeeping proteins (e.g., tubulin, GAPDH)

  • Cell fractionation control: Markers for subcellular compartments in localization studies

• Biological Context Controls:

  • Pathway inhibitor treatment: IKK complex inhibitors to reduce expected phosphorylation

  • Other pathway activators: Compare responses to different stimuli (IL-1β, LPS)

For multiplexed approaches (flow cytometry, multiplex Western blotting), include fluorescence minus one (FMO) controls and conduct spectral compensation to prevent false-positive signal detection .

How can I optimize Western blot protocols for detecting low-abundance phospho-IKBKG (S376)?

Detecting low-abundance phospho-IKBKG (S376) by Western blotting requires optimization at multiple steps:

• Sample Enrichment:

  • Increase protein loading (up to 50 μg per lane)

  • Consider immunoprecipitation with total IKBKG antibody before blotting with phospho-specific antibody

  • Use phosphoprotein enrichment columns to concentrate phosphorylated proteins

• Lysis Buffer Optimization:

  • Include potent phosphatase inhibitor cocktails (sodium fluoride, sodium orthovanadate, β-glycerophosphate)

  • Use RIPA or NP-40 based buffers with protease inhibitors

  • Maintain cold temperatures throughout lysate preparation

• Transfer Parameters:

  • Use PVDF membranes (0.45 μm pore size) for better protein retention

  • Implement wet transfer at low voltage (30V) overnight at 4°C

  • Add SDS (0.1%) to transfer buffer for higher molecular weight proteins

• Blocking and Antibody Incubation:

  • Block with 3-5% BSA in TBST (not milk, which contains phosphatases)

  • Extend primary antibody incubation to overnight at 4°C

  • Use antibody concentration at the higher end of recommended range (1 μg/mL)

  • Consider using signal enhancers like SignalBoost™ Immunoreaction Enhancer

• Detection System:

  • Use high-sensitivity ECL substrate systems for chemiluminescence

  • Consider longer exposure times, but watch for background

  • Use digital imaging systems with accumulation modes for weak signals

• Signal Amplification:

  • Implement tyramide signal amplification if conventional detection fails

  • Consider using biotin-streptavidin systems for additional signal enhancement

These optimizations have been demonstrated to improve detection of phospho-IKBKG in diverse cell types, including HeLa, NIH/3T3, and C6 cells .

What are common pitfalls in phospho-IKBKG (S376) detection and how can they be addressed?

Several technical challenges can compromise phospho-IKBKG (S376) detection. Here are common pitfalls and their solutions:

• Weak or Absent Signal:

  • Cause: Dephosphorylation during sample preparation or insufficient stimulation

  • Solution: Ensure rigorous phosphatase inhibition in all buffers; optimize stimulation conditions (e.g., TNF-α at 20 ng/mL for 30 minutes)

• Multiple Bands or Incorrect Molecular Weight:

  • Cause: Cross-reactivity, protein degradation, or unexpected post-translational modifications

  • Solution: Verify expected molecular weight (observed MW ~65 kDa despite calculated MW of 48 kDa) ; use fresh samples with protease inhibitors; validate with knockout/knockdown controls

• High Background:

  • Cause: Insufficient blocking, antibody concentration too high, or non-specific binding

  • Solution: Optimize blocking conditions (3-5% BSA in TBST); titrate antibody concentration; increase washing stringency

• Inconsistent Results Between Experiments:

  • Cause: Variation in cell culture conditions, stimulation protocols, or protein loading

  • Solution: Standardize cell culture conditions; implement strict timing protocols; normalize to total IKBKG and loading controls

• Poor Tissue Staining in IHC:

  • Cause: Inadequate antigen retrieval or overfixation

  • Solution: Optimize antigen retrieval methods (citrate buffer pH 6.0) ; test multiple fixation times

• Species Cross-Reactivity Issues:

  • Cause: The antibody may not recognize the target across species despite sequence homology

  • Solution: Verify antibody reactivity with the species of interest; consider species-specific antibodies

• Conflicting Data Between Techniques:

  • Cause: Different detection thresholds or epitope accessibility across methods

  • Solution: Validate findings using complementary techniques (Western blot, IHC, and flow cytometry); adjust protocols for each application

Each detection method may require specific optimization steps to achieve reliable phospho-IKBKG (S376) detection.

How can I distinguish between specific phospho-IKBKG (S376) signals and artifacts?

Distinguishing true phospho-IKBKG (S376) signals from artifacts requires rigorous validation approaches:

• Biological Validation:

  • Verify signal increases after appropriate stimulation (TNF-α, IL-1β)

  • Confirm signal reduction after pathway inhibition or phosphatase treatment

  • Demonstrate dose-dependent responses to stimuli

• Technical Validation:

  • Compare results using antibodies from different sources or clones

  • Use phospho-blocking peptides to confirm specificity

  • Test signal in IKBKG-deficient cells or tissues

• Pattern Recognition:

  • True signals show expected subcellular localization (primarily cytoplasmic for IKBKG)

  • Signal timing should match known pathway kinetics (rapid increase after stimulation)

  • Band pattern should be consistent across similar samples

• Quantitative Assessment:

  • Calculate signal-to-noise ratios to establish detection thresholds

  • Compare signal intensities to established positive controls

  • Evaluate correlation between phospho-signal and downstream functional outcomes

• Multiple Detection Methods:

  • Confirm key findings using orthogonal techniques (e.g., mass spectrometry)

  • Validate Western blot findings with immunofluorescence to assess cellular distribution

  • Consider proximity ligation assays to detect specific phosphorylation events in situ

Implementation of these validation strategies creates a framework for distinguishing genuine phosphorylation signals from technical artifacts .

What approaches can resolve contradictory results between different phospho-IKBKG (S376) antibodies?

When different phospho-IKBKG (S376) antibodies yield contradictory results, systematic troubleshooting is required:

• Epitope Mapping Analysis:

  • Determine the exact immunogen sequences used to generate each antibody

  • Assess whether antibodies recognize overlapping or distinct regions around S376

  • Test antibodies against phosphorylated and non-phosphorylated peptides in ELISA

• Antibody Characterization:

  • Compare antibody formats (monoclonal vs. polyclonal)

  • Assess host species and isotype differences

  • Review validation data provided by manufacturers

• Methodological Reconciliation:

  • Test antibodies side-by-side under identical conditions

  • Optimize protocols individually for each antibody

  • Evaluate sensitivity thresholds for each antibody

• Biological Verification:

  • Test antibodies in known positive control conditions (TNF-α stimulation)

  • Compare results after phosphatase treatment

  • Validate with genetic approaches (phospho-mutant S376A expression)

• Technical Comparison Matrix:

  Parameter	Antibody A	Antibody B	Resolution Strategy
  Immunogen	Synthetic peptide	Recombinant protein	Test with blocking peptides
  Host Species	Rabbit	Mouse	Use species-specific secondary antibodies
  Clonality	Polyclonal	Monoclonal	Evaluate batch consistency
  Optimal Dilution	1:100	1:1000	Titrate both antibodies
  Detection Method	Colorimetric	Fluorescent	Compare sensitivity limits

• Third-Method Validation:

  • Use mass spectrometry to directly detect and quantify phosphorylation at S376

  • Employ phospho-specific functional assays

  • Consider in vitro kinase assays with purified components

This systematic approach can reconcile contradictory results and establish which antibody provides the most accurate representation of IKBKG phosphorylation status .

How can phospho-IKBKG (S376) antibodies be used to study cross-talk between NF-κB and other signaling pathways?

Phospho-IKBKG (S376) antibodies offer powerful tools for investigating signaling cross-talk:

• Dual Pathway Activation Studies:

  • Simultaneously activate NF-κB and parallel pathways (MAPK, JAK/STAT, PI3K/Akt)

  • Monitor phospho-IKBKG (S376) levels during combined stimulation

  • Correlate IKBKG phosphorylation with activation markers from multiple pathways

• Inhibitor Matrix Approaches:

  • Apply specific inhibitors of various pathways in combination

  • Create inhibitor matrices to identify non-linear interactions affecting S376 phosphorylation

  • Use phospho-flow cytometry to assess pathway activities at single-cell resolution

• Time-Resolved Signaling Profiles:

  • Establish temporal relationships between IKBKG phosphorylation and other pathway activations

  • Determine whether S376 phosphorylation precedes or follows other signaling events

  • Identify potential feedback mechanisms regulating phosphorylation dynamics

• Multiparameter Phospho-Proteomics:

  • Combine phospho-IKBKG antibodies with broader phospho-proteomic analyses

  • Create phosphorylation networks to map pathway interconnections

  • Identify novel regulatory relationships involving IKBKG

• Stress Response Integration:

  • Examine how cellular stresses (oxidative, ER, genotoxic) influence S376 phosphorylation

  • Determine whether S376 phosphorylation serves as an integration point for multiple stressors

  • Study how metabolic status affects NF-κB activation through IKBKG phosphorylation

These approaches have revealed that TNF-α-induced IKBKG phosphorylation involves coordination between IKK complex activation and other pathways, providing insight into the complex regulation of inflammatory responses .

What is known about the kinases responsible for IKBKG S376 phosphorylation and methods to study them?

The specific kinases mediating IKBKG S376 phosphorylation remain incompletely characterized, though several approaches can identify and study these enzymes:

• Candidate Kinase Screening:

  • Several kinases have been implicated in IKBKG phosphorylation, including:

    • IKKβ (auto-phosphorylation within the IKK complex)

    • ATM/ATR (DNA damage response kinases)

    • TBK1/IKKε (non-canonical IKK family members)

  • Systematically inhibit or deplete these kinases and assess effects on S376 phosphorylation

• Kinase Assay Approaches:

  • Perform in vitro kinase assays using purified candidate kinases and IKBKG as substrate

  • Implement kinase substrate tracking and elucidation (KESTREL) methods

  • Use phosphorylation-specific mass spectrometry to confirm S376 phosphorylation

• Chemical Genetics:

  • Apply analog-sensitive kinase technology to identify direct substrates

  • Use kinase inhibitor panels to narrow down candidate kinases

  • Implement CRISPR screens targeting kinome members

• Computational Prediction:

  • S376 sits within a sequence context (pSer-Pro) that may represent a proline-directed kinase motif

  • Bioinformatic tools suggest potential kinases based on sequence context

  • Molecular modeling can predict kinase-substrate interactions

• Context-Dependent Regulation:

  • Different stimuli (TNF-α, IL-1β, genotoxic stress) may utilize distinct kinases for S376 phosphorylation

  • Cell type-specific kinase expression patterns may influence which enzyme predominates

  • Subcellular localization of IKBKG may determine accessible kinases

Research suggests that TNF-α-induced phosphorylation likely involves IKKβ through a conformational change in the IKK complex, while DNA damage-induced phosphorylation may involve ATM kinase .

How can phospho-IKBKG (S376) be used as a biomarker in disease models and potentially in clinical samples?

Phospho-IKBKG (S376) shows promise as a biomarker in various disease contexts:

• Inflammatory Disease Models:

  • Monitor phospho-IKBKG levels in tissues from inflammatory disease models

  • Correlate phosphorylation with disease severity and progression

  • Evaluate therapeutic responses through changes in phosphorylation status

• Cancer Research Applications:

  • Assess phospho-IKBKG in tumor samples to determine NF-κB pathway activation

  • Correlate with treatment resistance phenotypes

  • Use as a companion biomarker for therapies targeting NF-κB signaling

• Methodological Considerations for Clinical Translation:

  • Establish robust IHC protocols for FFPE tissue samples

  • Develop quantitative scoring systems for phospho-IKBKG levels

  • Create standardized controls for inter-laboratory reproducibility

• Single-Cell Analysis:

  • Apply phospho-flow cytometry to detect heterogeneity in clinical samples

  • Identify specific cell populations with elevated phospho-IKBKG

  • Correlate with other activation markers at single-cell resolution

• Longitudinal Monitoring:

  • Establish baseline phosphorylation in healthy tissues

  • Monitor changes during disease progression

  • Track therapeutic responses through serial sampling

• Multiparameter Disease Profiling:

  • Combine phospho-IKBKG with other NF-κB pathway markers

  • Create comprehensive signaling profiles for patient stratification

  • Develop predictive models based on pathway activation patterns

While research applications are well-established, clinical implementation requires additional validation of preanalytical variables affecting phosphorylation status, standardization of detection methods, and correlation with clinical outcomes .

How does IKBKG S376 phosphorylation compare with other post-translational modifications of IKBKG?

IKBKG undergoes multiple post-translational modifications (PTMs) that collectively regulate its function:

• Comparative Profile of Major IKBKG PTMs:

  Modification	Site(s)	Function	Relationship to S376 Phosphorylation
  Phosphorylation	S376	Regulates NF-κB activation	Primary focus of this FAQ
  Phosphorylation	S68	Attenuates N-terminal homodimerization	May work in coordination with S376 phosphorylation
  Ubiquitination (K63-linked)	K285, K399	Facilitates signaling interactions	May create binding platforms for kinases that phosphorylate S376
  Ubiquitination (Mono)	K277, K309	Promotes nuclear export	May affect subcellular localization of phosphorylated IKBKG
  Ubiquitination (K27-linked)	Multiple sites	Involved in antiviral responses	May represent parallel regulatory mechanism
  Linear Ubiquitination	K111, K143, etc.	Key role in NF-κB activation	May be prerequisite for S376 phosphorylation

• Temporal Relationships:

  • Ubiquitination events often precede phosphorylation

  • S376 phosphorylation may require prior modifications to create appropriate structural context

  • Different stimuli may trigger distinct PTM sequences

• Functional Interplay:

  • S376 phosphorylation may influence susceptibility to subsequent ubiquitination

  • Certain ubiquitination patterns may create docking sites for S376 kinases

  • Combined PTMs likely create a "code" determining IKBKG functional outcomes

• Methodological Approaches to Study PTM Crosstalk:

  • Sequential immunoprecipitation with PTM-specific antibodies

  • Mass spectrometry to identify co-occurring modifications

  • Generation of mutants affecting specific modifications

• Stimulus-Specific Patterns:

  • TNF-α stimulation triggers both ubiquitination and S376 phosphorylation

  • DNA damage response may favor different PTM combinations

  • Pathogen recognition receptor signaling creates unique PTM profiles

Understanding the interplay between these modifications provides deeper insight into IKBKG regulation and may reveal new therapeutic opportunities .

What emerging techniques are enhancing our ability to study IKBKG phosphorylation dynamics?

Several cutting-edge methodologies are transforming research on IKBKG phosphorylation:

• Live-Cell Phosphorylation Sensors:

  • Genetically encoded FRET-based biosensors for real-time phosphorylation monitoring

  • Split luciferase systems reporting phosphorylation events

  • Fluorescent phospho-binding domain reporters

• Single-Molecule Imaging:

  • Super-resolution microscopy to visualize individual IKBKG molecules

  • Single-particle tracking to follow phosphorylated IKBKG dynamics

  • Correlative light-electron microscopy for structural context

• Spatial Proteomics:

  • Cellular compartment-specific phospho-proteomics

  • Subcellular fractionation coupled with phospho-specific detection

  • Proximity labeling of phospho-IKBKG interaction partners

• Advanced Mass Spectrometry:

  • Targeted parallel reaction monitoring for precise phospho-site quantification

  • Top-down proteomics to capture full combinatorial PTM landscapes

  • Crosslinking mass spectrometry to identify conformational changes upon phosphorylation

• Optogenetic and Chemogenetic Approaches:

  • Light-controllable kinase systems to induce S376 phosphorylation with spatiotemporal precision

  • Chemically induced proximity systems to trigger phosphorylation events

  • Engineered allosteric switches to study phosphorylation consequences

• In Situ Structural Biology:

  • Cryo-electron tomography to visualize IKK complexes in cellular context

  • Integrative structural modeling incorporating phosphorylation states

  • Hydrogen-deuterium exchange mass spectrometry to detect conformational changes

These emerging technologies are enabling unprecedented insights into the dynamics, spatial organization, and functional consequences of IKBKG phosphorylation events .

How might targeting IKBKG phosphorylation inform therapeutic development strategies?

The central role of IKBKG phosphorylation in NF-κB signaling suggests several therapeutic development opportunities:

• Direct Targeting Approaches:

  • Development of phosphorylation-site specific inhibitors blocking S376 phosphorylation

  • Peptide-based inhibitors mimicking the S376 region to compete for kinase binding

  • Stabilization of non-phosphorylated conformations through allosteric modulators

• Kinase-Directed Strategies:

  • Identification and inhibition of specific kinases responsible for S376 phosphorylation

  • Development of degraders (PROTACs) for these kinases

  • Creation of substrate-selective kinase inhibitors to specifically block IKBKG phosphorylation

• Phosphatase Enhancement:

  • Identification of phosphatases that dephosphorylate S376

  • Small molecule activators of these phosphatases

  • Targeted phosphatase recruitment strategies

• Context-Specific Modulation:

  • Targeting disease-specific mechanisms that enhance S376 phosphorylation

  • Tissue-selective delivery of phosphorylation modulators

  • Pathway-selective interventions that spare beneficial NF-κB functions

• Biomarker Applications:

  • Use of phospho-IKBKG (S376) as a companion diagnostic

  • Patient stratification based on baseline phosphorylation status

  • Pharmacodynamic marker for NF-κB pathway inhibitors

• Combination Therapy Rationales:

  • Targeting IKBKG phosphorylation alongside other NF-κB regulatory mechanisms

  • Combining with ubiquitination modulators for synergistic effects

  • Sequence-specific therapy based on PTM dependencies

These approaches may be particularly relevant in inflammatory disorders, certain cancers, and autoimmune conditions where aberrant NF-κB signaling contributes to pathology .