Phospho-NFKB1 (Ser337) Antibody

What is NFKB1 and what role does phosphorylation at Ser337 play?

NFKB1 encodes a 105 kDa protein (p105) that undergoes processing by the 26S proteasome to produce a 50 kDa protein (p50). The p50 subunit serves as a DNA binding component of the NF-κB transcription factor complex, which regulates numerous biological processes including inflammation, immunity, cell differentiation, and apoptosis .

Phosphorylation at Ser337 is specifically critical for the DNA binding function of the p50 subunit. Research has demonstrated that this site lies within a Protein Kinase A (PKA) consensus sequence and PKA has been confirmed to phosphorylate p50 at Ser337 both in vitro and in vivo . Mutation studies have shown that substitution of Ser337 with negatively charged aspartic acid fails to restore DNA binding, while substitution with threonine (another potential phospho-acceptor) retains DNA binding capability .

What are the primary applications for Phospho-NFKB1 (Ser337) antibodies?

Phospho-NFKB1 (Ser337) antibodies are valuable tools for several experimental applications:

These antibodies are particularly useful for monitoring changes in NFKB1 phosphorylation status following cellular treatments with various stimuli, inhibitors, or activators .

How should I validate the specificity of Phospho-NFKB1 (Ser337) antibodies?

Validating antibody specificity is crucial for reliable experimental outcomes. The following approaches are recommended:

• Peptide competition assays: Compare antibody reactivity with phosphorylated versus non-phosphorylated peptides. Highly specific antibodies show significantly higher reactivity with phospho-peptides .

• Treatment controls: Utilize known activators of the NF-κB pathway such as TNF-α (20 ng/ml) with Calyculin A (100 nM) for 10-30 minutes to induce phosphorylation . This treatment dramatically increases the phospho-signal in Western blots compared to untreated controls.

• Cell line verification: Use established cell lines like HeLa, NIH/3T3, or C6 cells, which show robust phosphorylation responses .

• Phosphatase treatment: Treat one sample with lambda phosphatase to remove the phosphate group; a specific phospho-antibody should show diminished signal in the dephosphorylated sample.

What are the recommended storage and handling conditions?

For optimal antibody performance and longevity, follow these guidelines:

• Store at -20°C for up to one year from the receipt date

• Avoid repeated freeze-thaw cycles to prevent protein denaturation

• Most formulations contain preservatives such as sodium azide (0.02-0.05%) and stabilizers like glycerol (50%) and BSA (0.5-1%)

• Upon receipt, aliquot the antibody to minimize freeze-thaw cycles

• When working with the antibody, keep it on ice or at 4°C

• Ship with blue ice or polar packs at 2-8°C

How does phosphorylation of Ser337 mechanistically influence DNA binding of NF-κB p50?

Phosphorylation of Ser337 critically regulates the DNA binding function of the p50 subunit through several mechanisms:

• Structural effects: Phosphorylation introduces a negative charge that alters protein conformation in a manner that enhances DNA binding. Unlike many phosphorylation events, substitution with a negatively charged amino acid (aspartic acid) cannot mimic this effect, suggesting that the phosphorylation induces a specific structural change beyond simple charge introduction .

• DNA binding kinetics: In vitro phosphorylation of Ser337 by PKA dramatically enhances the DNA binding ability of p50, indicating that this modification increases the affinity of p50 for κB sites in target gene promoters .

• Dimerization effects: While Ser337 phosphorylation primarily affects DNA binding, it may also influence dimer stability. This is suggested by research on the nearby residue Ser340, which is critical for p50 homodimer formation. The proximity of these sites raises the possibility of cooperative effects between phosphorylation and dimerization .

• Context-dependent regulation: Unlike Ser328 phosphorylation (which selectively affects binding to κB-sites with specific nucleotide sequences), Ser337 phosphorylation appears to be a more general enhancer of DNA binding across various κB sites .

What experimental approaches can detect changes in Ser337 phosphorylation following cell stimulation?

To effectively monitor changes in Ser337 phosphorylation, researchers should consider these methodological approaches:

• Cell-Based Phosphorylation ELISA:

  • Plate cells directly in 96-well format (30,000 HeLa cells/well is optimal)

  • Treat with appropriate stimuli (e.g., TNF-α at 20 ng/ml for 5-10 minutes)

  • Fix cells with 4% formaldehyde

  • Perform ELISA using both phospho-specific and total NFKB1 antibodies

  • Normalize to cell number using GAPDH antibody or Crystal Violet staining

• Western Blot Analysis:

  • Treat cells with activators (TNF-α, 20 ng/ml) and phosphatase inhibitors (Calyculin A, 100 nM) for 10-30 minutes

  • Extract proteins using phosphatase inhibitor-containing buffers

  • Run SDS-PAGE and transfer to membrane

  • Probe with Phospho-NFKB1 (Ser337) antibody (1:500-1:2000 dilution)

  • Re-probe with total NFKB1 antibody for normalization

  • Look for bands at approximately 50 kDa (p50) and 105-120 kDa (p105)

• Proximity Ligation Assay (PLA):

  • Using antibody pairs that target both phosphorylated Ser337 and total NFKB1

  • This approach allows visualization of phosphorylation events at the single-molecule level within intact cells

  • Antibody pairs are designed to generate a signal only when both antibodies bind in close proximity, confirming specific phosphorylation

How do different cellular stimuli affect NFKB1 Ser337 phosphorylation?

Different stimuli have distinct effects on Ser337 phosphorylation of NFKB1:

Temporal dynamics of phosphorylation are important - many stimuli induce rapid but transient phosphorylation, necessitating careful time-course experiments to capture optimal phosphorylation windows .

What is the relationship between Ser337 phosphorylation and other post-translational modifications of NFKB1?

NFKB1 undergoes multiple post-translational modifications that can interact with Ser337 phosphorylation:

• Relationship with other phosphorylation sites: While Ser337 phosphorylation primarily regulates DNA binding, phosphorylation at Ser328 (by Chk1 kinase) selectively affects binding to specific κB sequences based on nucleotide composition . The interplay between these phosphorylation events may determine gene-specific transcriptional outcomes.

• Interplay with p105 processing: Phosphorylation events in the C-terminal region of p105 control its processing to p50 by the proteasome. How Ser337 phosphorylation in the resulting p50 coordinates with these upstream processing events remains an important area for investigation .

• Potential crosstalk with Ser340: Research suggests that Ser340 is critical for p50 homodimer formation, and mutation of this residue recapitulates the nfkb1−/− phenotype in a hepatocellular carcinoma model . The proximity of Ser337 and Ser340 suggests possible functional interactions between these sites.

• Temporal sequence of modifications: The order of post-translational modifications can significantly impact NFKB1 function. Current evidence suggests that phosphorylation at Ser337 is an early event that enables DNA binding, while other modifications may subsequently fine-tune transcriptional activity .

How can I design functional experiments to assess the biological significance of Ser337 phosphorylation?

To explore the functional significance of Ser337 phosphorylation:

• Site-directed mutagenesis approach:

  • Generate S337A (non-phosphorylatable) and S337T (phospho-mimetic) mutations

  • Express these mutants in appropriate cell lines

  • Assess DNA binding via EMSA (Electrophoretic Mobility Shift Assay)

  • Measure transcriptional activity using reporter gene assays

  • Compare nuclear localization and protein stability between variants

• Pharmacological approach:

  • Use specific PKA inhibitors to block Ser337 phosphorylation

  • Compare with effects of inhibitors targeting other kinases

  • Monitor downstream gene expression changes

  • Assess effects on biological processes like inflammation or apoptosis

• CRISPR/Cas9 genome editing:

  • Generate cell lines or mouse models with S337A or S337T knock-in mutations

  • Compare phenotypes with wild-type and complete NFKB1 knockout

  • Challenge with inflammatory stimuli or DNA damage

  • Assess transcriptional responses using RNA-seq or targeted qPCR

  • This approach can reveal gene-specific effects of Ser337 phosphorylation

• Phosphatase identification:

  • Identify phosphatases that dephosphorylate Ser337

  • Manipulate phosphatase activity to modulate phosphorylation levels

  • This can reveal dynamic regulation mechanisms

What controls should be included when using Phospho-NFKB1 (Ser337) antibodies?

Proper experimental controls are essential:

• Positive controls:

  • Cells treated with TNF-α (20 ng/ml) for 5-10 minutes

  • Cells treated with Calyculin A (100 nM) for 30 minutes after serum starvation

  • These treatments induce robust Ser337 phosphorylation

• Negative controls:

  • Untreated cells (baseline phosphorylation)

  • Secondary antibody alone (background signal)

  • Phosphatase-treated lysates (to confirm phospho-specificity)

• Normalization controls:

  • Total NFKB1 antibody (for phosphorylation level normalization)

  • GAPDH antibody (for loading control and cell number normalization)

  • Crystal Violet staining (for cell-based assays)

• Peptide competition:

  • Pre-incubation of antibody with phospho-peptide versus non-phospho-peptide

  • Should show selective blocking with phospho-peptide only

How do I troubleshoot weak or non-specific signal issues?

When encountering signal problems with Phospho-NFKB1 (Ser337) antibodies:

• Weak signal troubleshooting:

  • Ensure cells were properly stimulated (TNF-α or Calyculin A)

  • Increase antibody concentration (try 1:500 instead of 1:2000 for Western blot)

  • Extend primary antibody incubation (overnight at 4°C)

  • Use enhanced detection systems (high-sensitivity ECL)

  • Add phosphatase inhibitors to all buffers

  • Reduce washing stringency

• Non-specific signal troubleshooting:

  • Verify antibody specificity through peptide competition

  • Increase blocking concentration (5% BSA instead of 3%)

  • Increase wash stringency

  • Reduce primary antibody concentration

  • Use freshly prepared buffers

  • Consider alternative antibody vendors/lots

• High background solutions:

  • Extend blocking time

  • Use alternative blocking agents (5% BSA often works better than milk for phospho-antibodies)

  • Include 0.1% Tween-20 in all wash and antibody dilution buffers

  • Reduce exposure time for ECL detection

How can I quantitatively analyze NFKB1 Ser337 phosphorylation changes?

For quantitative analysis of phosphorylation changes:

• Western blot densitometry:

  • Always normalize phospho-NFKB1 signal to total NFKB1

  • Use linear range of detection for accurate quantification

  • Include multiple exposure times

  • Apply statistical analysis across multiple independent experiments

• Cell-based ELISA quantification:

  • Set up a minimum of triplicate wells for each condition

  • Normalize phospho-signal to total protein or cell number

  • Use GAPDH as internal control

  • Apply appropriate statistical tests (t-test, ANOVA)

• Flow cytometry:

  • For single-cell quantification of phosphorylation levels

  • Combine with markers for specific cell populations

  • Provides distribution data rather than just population averages

• Proximity Ligation Assay (PLA) quantification:

  • Count PLA signal dots per cell using microscopy

  • Provides quantitative spatial information about phosphorylation events

  • Can be combined with other markers for contextual information

How does NFKB1 Ser337 phosphorylation impact gene-specific transcriptional regulation?

The impact of Ser337 phosphorylation on gene-specific regulation is complex:

• DNA binding versus transcriptional activity: While Ser337 phosphorylation primarily enhances DNA binding capability , the relationship between enhanced binding and transcriptional output varies between target genes.

• Comparison with other phosphorylation sites: Unlike Ser328 phosphorylation, which selectively affects binding to κB-sites with specific nucleotide compositions (cytosine at the -1 position), Ser337 phosphorylation appears to be a more general enhancer of DNA binding . This suggests it may broadly activate NF-κB target genes rather than selectively regulate specific gene subsets.

• Dimer-specific effects: Phosphorylation of Ser337 may differentially impact various NF-κB dimers (p50/p65 heterodimers versus p50 homodimers). Since p50 homodimers often repress gene expression while p50/p65 heterodimers activate expression, understanding dimer-specific effects is critical .

• Temporal dynamics: The timing of Ser337 phosphorylation in relation to stimulus exposure and other NF-κB modifications likely determines which target genes are activated and for how long.

What is the role of Ser337 phosphorylation in pathological contexts?

NFKB1 Ser337 phosphorylation has important implications in several disease contexts:

• Inflammatory disorders: Enhanced NFKB1 Ser337 phosphorylation may contribute to hyperactivation of NF-κB signaling in chronic inflammatory conditions.

• Cancer: Aberrant regulation of Ser337 phosphorylation could impact NF-κB's role in cancer development and progression. The nearby residue Ser340 has been specifically implicated in hepatocellular carcinoma models .

• Immune dysfunction: Given NF-κB's central role in immune responses, dysregulated Ser337 phosphorylation could contribute to immunodeficiency or autoimmunity.

• Therapeutic targeting: Understanding the kinases and phosphatases that regulate Ser337 phosphorylation could provide novel therapeutic targets for modulating NF-κB activity in a more selective manner than current approaches.

How do species differences affect NFKB1 Ser337 phosphorylation studies?

When conducting cross-species research on NFKB1 Ser337 phosphorylation:

• Conservation: The Ser337 phosphorylation site is conserved across human, mouse, and rat species, making it possible to use the same phospho-specific antibodies across these species .

• Peptide sequence context: The peptide sequence surrounding Ser337 (R-K-S-D-L) is highly conserved between human, mouse, and rat NFKB1 , explaining the cross-reactivity of many commercially available antibodies.

• Regulatory differences: Despite sequence conservation, the regulation of Ser337 phosphorylation (including responsible kinases and phosphatases) may differ between species.

• Experimental validation: When using phospho-specific antibodies in a new species, validation experiments (such as stimulus-induced phosphorylation) should be performed to confirm antibody performance in that species.

• Model selection: For translational research, consider which animal model best recapitulates the regulatory mechanisms of Ser337 phosphorylation relevant to human disease.