Phospho-NCF1 (Ser345) Antibody

What is Phospho-NCF1 (Ser345) Antibody and what specific epitope does it recognize?

Phospho-NCF1 (Ser345) Antibody is a rabbit polyclonal antibody that specifically detects endogenous levels of p47-phox (NCF1) protein only when phosphorylated at serine 345. The antibody is typically generated using synthetic phosphopeptides derived from human NCF1 around the phosphorylation site of Ser345 (amino acid range 311-360) . This specificity allows researchers to distinguish between the phosphorylated and non-phosphorylated forms of NCF1, making it valuable for studying activation states of the NADPH oxidase complex.

The antibody recognizes the amino acid sequence around phosphorylation site of Serine 345 (P-Q-S(p)-P-G) derived from Human NCF1 . Proper validation of this antibody involves demonstrating reduced or absent signal following phosphatase treatment of samples, confirming its phospho-specificity.

What are the validated research applications for Phospho-NCF1 (Ser345) Antibody?

The Phospho-NCF1 (Ser345) Antibody has been validated for multiple research applications:

For optimal results, researchers should perform dilution optimization for their specific experimental conditions and sample types. The antibody has been successfully used to detect phosphorylated NCF1 in various cell types, including neutrophils and RAW264.7 macrophages .

What is the biological function of NCF1/p47-phox in cellular processes?

NCF1 (Neutrophil Cytosol Factor 1), also known as p47-phox, is a critical component of the NADPH oxidase complex. In conjunction with NCF2 and membrane-bound cytochrome b558, NCF1 is required for activation of the latent NADPH oxidase, which is necessary for superoxide production .

The primary functions of NCF1 include:

• Serving as an organizer protein (also called NOXO2) for NADPH oxidase assembly

• Mediating translocation of cytosolic components to the membrane during oxidase activation

• Facilitating innate immune responses, particularly in neutrophils and macrophages

• Participating in pathogen clearance through reactive oxygen species production

Phosphorylation of NCF1 at Ser345 represents a key regulatory mechanism that enhances NADPH oxidase activity . Mutations or dysfunction in NCF1 are associated with chronic granulomatous disease, characterized by recurrent infections due to impaired phagocyte superoxide production .

What is the recommended storage and handling protocol for Phospho-NCF1 (Ser345) Antibody?

To maintain optimal activity of Phospho-NCF1 (Ser345) Antibody, the following storage and handling protocols are recommended:

• Store at -20°C for up to 1 year from the date of receipt

• For frequent use and short-term storage, aliquot and keep at 4°C for up to one month

• Avoid repeated freeze-thaw cycles to prevent antibody degradation

• The antibody is typically supplied in PBS containing 50% glycerol, 0.5% BSA and 0.02% sodium azide

• Always centrifuge the antibody vial briefly before opening to collect solution at the bottom of the tube

• When diluting for experimental use, use fresh, cold buffer and prepare only the amount needed for immediate use

Proper storage and handling are critical for maintaining antibody performance across multiple experiments and extending its shelf life.

What species reactivity has been confirmed for Phospho-NCF1 (Ser345) Antibody?

The species reactivity of Phospho-NCF1 (Ser345) Antibody has been experimentally validated for:

When applying this antibody to species not listed above, researchers should perform proper validation to confirm cross-reactivity before proceeding with full-scale experiments. The high sequence conservation of the phosphorylation site region across mammalian species suggests potential cross-reactivity with other mammals, though this requires experimental verification .

How does phosphorylation of NCF1 at Ser345 regulate NADPH oxidase activity and superoxide production?

Phosphorylation of NCF1 at Ser345 serves as a key regulatory mechanism for NADPH oxidase activation and subsequent superoxide production. This post-translational modification induces conformational changes in NCF1 that facilitate:

• Enhanced interaction with other NADPH oxidase components (p22phox, p67phox)

• Increased membrane translocation of the cytosolic oxidase components

• Priming of NADPH oxidase for full activation following secondary stimuli

Research indicates that Ser345 phosphorylation is mediated by various signaling pathways including p38 MAPK . A study by Bhowmick et al. demonstrated that N-SLIT2 significantly increases phosphorylation of the Ser345 residue of NCF1 in neutrophils, with densitometry analysis showing a ratio of phospho-NCF1/NCF1 with p=0.0002 when comparing vehicle vs N-SLIT2 treatment .

Importantly, this phosphorylation represents a priming mechanism that enhances reactive oxygen species (ROS) production without necessarily triggering oxidative burst immediately, suggesting its role in preparing neutrophils for enhanced respiratory burst upon subsequent stimulation.

What signaling pathways have been implicated in regulating NCF1 phosphorylation at Ser345?

Several signaling pathways have been identified as regulators of NCF1 Ser345 phosphorylation:

Recent research by Bhowmick et al. demonstrated that N-SLIT2 increases phosphorylation of NCF1 at Ser345 in both neutrophils and RAW264.7 macrophages. The effect was specific to the bioactive N-SLIT2, as the N-SLIT2ΔD2 variant did not induce this phosphorylation (p=0.0004 when comparing N-SLIT2 vs N-SLIT2ΔD2) .

Understanding these regulatory pathways provides potential therapeutic targets for modulating oxidative burst in inflammatory and infectious diseases.

What experimental controls should be included when using Phospho-NCF1 (Ser345) Antibody in Western blot analysis?

To ensure reliable and interpretable results when using Phospho-NCF1 (Ser345) Antibody in Western blot experiments, the following controls should be included:

• Phosphatase treatment control: Treat duplicate samples with lambda phosphatase to demonstrate the phospho-specificity of the antibody signal

• Total NCF1 detection: Run parallel blots or strip and reprobe with an antibody against total NCF1 to normalize phosphorylation levels

• Positive control: Include samples known to have high levels of NCF1 Ser345 phosphorylation (e.g., neutrophils treated with N-SLIT2 or other activators)

• Negative control: Include samples with minimal NCF1 Ser345 phosphorylation (e.g., resting cells)

• Loading control: Probe for housekeeping proteins to ensure equal protein loading across lanes

• Blocking peptide control: When validating the antibody, include samples where the antibody is pre-incubated with the phosphopeptide immunogen

For quantitative analysis, densitometry should be performed to calculate the ratio of phospho-NCF1/total NCF1, as demonstrated in studies examining N-SLIT2 effects on neutrophils .

How does the NCF1 variant p.R90H influence autoimmunity through pDC activation?

The NCF1 variant p.R90H has been identified as a genetic risk factor that aggravates autoimmunity through its effects on plasmacytoid dendritic cells (pDCs). Research has uncovered several mechanisms:

• Reduced oxidative stress: pDCs carrying the NCF1 p.R90H variant (A/A genotype) show downregulation of oxidative stress pathways

• Enhanced TLR pathway activation: RNA profiling of A/A pDCs demonstrated excessive activation of Toll-like receptor pathways

• Increased phosphorylation of key signaling molecules: The A/A pDCs exhibit elevated phosphorylation levels of IRF7 and NF-κB under stimulation with R848 or CpG

This variant affects a different region of NCF1 than the Ser345 phosphorylation site, suggesting complex regulatory mechanisms within the protein. While studying NCF1 phosphorylation at Ser345, researchers should consider the genetic background of their experimental system, as variants like p.R90H may influence NADPH oxidase activity independently of Ser345 phosphorylation status.

What is the relationship between N-SLIT2 and NCF1 Ser345 phosphorylation in neutrophil function?

Recent research has identified N-SLIT2 as a significant modulator of NCF1 Ser345 phosphorylation and neutrophil function. The key findings include:

• N-SLIT2 significantly increases phosphorylation of NCF1 at Ser345 in human neutrophils (p=0.0002 compared to vehicle control)

• This effect is specific to bioactive N-SLIT2, as N-SLIT2ΔD2 (deletion variant) does not induce similar phosphorylation (p=0.0004 when comparing N-SLIT2 vs N-SLIT2ΔD2)

• The N-SLIT2-induced phosphorylation of NCF1 is associated with enhanced innate immunity against infections

• The effect appears to be mediated through p38 MAPK signaling pathway rather than PKC activation

• Similar effects were observed in RAW264.7 macrophages, suggesting a conserved mechanism across different phagocytic cells

This relationship between N-SLIT2 and NCF1 phosphorylation reveals a novel mechanism by which chemorepellents can bolster innate immunity through modulation of NADPH oxidase activation pathways.

How can I optimize immunohistochemistry protocols for detecting phosphorylated NCF1 in tissue samples?

Optimizing immunohistochemistry (IHC) protocols for Phospho-NCF1 (Ser345) detection requires careful attention to several factors:

• Fixation and antigen retrieval:

  • Use 10% neutral buffered formalin for tissue fixation

  • Test multiple antigen retrieval methods (citrate buffer pH 6.0, EDTA buffer pH 9.0)

  • Heat-induced epitope retrieval is generally preferred for phospho-epitopes

• Blocking and antibody dilution optimization:

  • Use 5-10% normal serum from the species of secondary antibody

  • Include phosphatase inhibitors in blocking solutions

  • Test a range of antibody dilutions (1:50-1:300 recommended)

  • Incubate primary antibody overnight at 4°C

• Signal detection and specificity controls:

  • Use phospho-blocking peptide as negative control

  • Include tissues known to express high levels of phospho-NCF1 as positive controls

  • Consider signal amplification systems for low abundance phospho-proteins

Immunohistochemistry analysis using Phospho-NCF1 (Ser345) antibody has been successfully performed on paraffin-embedded human tissues, with specific staining that can be blocked with the phospho-peptide used for immunization .

What methodological approaches can be used to study the temporal dynamics of NCF1 Ser345 phosphorylation?

Studying the temporal dynamics of NCF1 Ser345 phosphorylation requires methods that can capture rapid changes in phosphorylation status:

• Time-course experiments with Western blotting:

  • Stimulate cells for various durations (e.g., 0, 5, 15, 30, 60 min)

  • Rapidly lyse cells in buffer containing phosphatase inhibitors

  • Perform Western blotting with Phospho-NCF1 (Ser345) antibody

  • Normalize to total NCF1 levels

• Live-cell imaging approaches:

  • Generate phospho-specific biosensors based on FRET technology

  • Combine with fluorescently tagged NCF1 to track localization and phosphorylation

  • Image at high temporal resolution (seconds to minutes)

• Phosphoproteomics:

  • Use SILAC or TMT labeling combined with mass spectrometry

  • Enrich for phosphopeptides using titanium dioxide or IMAC

  • Quantify Ser345-containing peptides across time points

• Flow cytometry for high-throughput analysis:

  • Fix and permeabilize cells at different time points

  • Stain with Phospho-NCF1 (Ser345) antibody and fluorescent secondary

  • Analyze by flow cytometry for population-level changes

Research has shown that N-SLIT2 induces rapid phosphorylation of NCF1 at Ser345 within 15 minutes of exposure, highlighting the importance of capturing early time points in signaling studies .