Phospho-LMNA (S392) Antibody

What is Phospho-LMNA (S392) Antibody and what epitope does it recognize?

Phospho-LMNA (S392) Antibody is a polyclonal antibody that specifically recognizes Lamin A/C protein when phosphorylated at the serine 392 residue. It is typically raised in rabbits against a synthesized peptide derived from human Lamin A/C containing the phosphorylated S392 site . The antibody has been designed to detect this specific post-translational modification without cross-reactivity to the non-phosphorylated form, making it valuable for studying the functional differences between phosphorylated and non-phosphorylated LMNA . The specificity for the phosphorylated epitope can be confirmed through phosphatase treatment controls that demonstrate signal reduction after dephosphorylation .

What is the biochemical composition and storage requirements for Phospho-LMNA (S392) Antibody?

Phospho-LMNA (S392) Antibody is typically supplied as a liquid formulation in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide as a preservative . The antibody is usually purified through affinity chromatography using the epitope-specific immunogen to ensure high specificity . For optimal stability and activity retention, the antibody should be stored at -20°C or -80°C immediately upon receipt . Researchers should avoid repeated freeze-thaw cycles as this can compromise antibody integrity and performance in experimental applications . For long-term studies, aliquoting the antibody before freezing is recommended to maintain consistent experimental results throughout the research project.

What species reactivity has been confirmed for Phospho-LMNA (S392) Antibody?

Phospho-LMNA (S392) Antibody has been validated for reactivity with human, mouse, and rat samples . This cross-species reactivity is due to the high conservation of the S392 phosphorylation site and surrounding amino acid sequence in the LMNA protein across these mammalian species. The antibody's ability to recognize this epitope across multiple species makes it particularly valuable for comparative studies and for establishing animal models of human diseases associated with LMNA phosphorylation . When planning experiments with other species not listed in the validated reactivity profile, researchers should perform preliminary validation tests using positive control samples to confirm cross-reactivity before proceeding with full-scale experiments.

What are the validated applications for Phospho-LMNA (S392) Antibody and their recommended dilutions?

Phospho-LMNA (S392) Antibody has been validated for multiple experimental applications with the following recommended dilutions:

These applications enable researchers to investigate the expression, localization, and dynamics of phosphorylated LMNA (S392) in various experimental contexts . For optimal results, researchers should optimize the dilution for their specific experimental conditions, sample types, and detection systems. Validation experiments in LMNA knockout cell lines have confirmed the specificity of the antibody signal in these applications .

How should positive and negative controls be designed for Phospho-LMNA (S392) Antibody experiments?

For rigorous experimental design with Phospho-LMNA (S392) Antibody, appropriate controls are essential:

Positive Controls:

• Cell lines treated with agents that activate MAP kinase signaling, such as growth factors or oncogenic Ras expression, which increase S392 phosphorylation

• Cell populations enriched in G2/M phase, when LMNA phosphorylation is elevated

• Phospho-mimetic LMNA constructs (S392D) expressed in LMNA-knockout cells

Negative Controls:

• LMNA knockout (LMNA-/-) cell lines to verify signal specificity

• Phosphatase-treated samples to confirm phosphorylation-dependent recognition

• Non-phosphorylatable LMNA mutants (S392A) expressed in LMNA-knockout cells

• Pre-incubation of the antibody with phosphorylated peptide immunogen for blocking experiments

These controls help confirm the specificity of the antibody and validate experimental findings related to LMNA S392 phosphorylation state . When interpreting results, comparison between phosphorylated and non-phosphorylated LMNA antibodies can provide complementary data on the distribution and dynamics of different LMNA forms.

What is the most effective protocol for immunofluorescence using Phospho-LMNA (S392) Antibody?

For optimal immunofluorescence detection of phosphorylated LMNA (S392), the following protocol is recommended:

• Fixation and Permeabilization:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

  • Critical: Avoid methanol fixation as it can affect phospho-epitopes

• Blocking and Antibody Incubation:

  • Block with 5% BSA in PBS for 1 hour at room temperature

  • Incubate with Phospho-LMNA (S392) Antibody at 1:200-1:1000 dilution in blocking buffer overnight at 4°C

  • Wash 3 times with PBS containing 0.1% Tween-20

• Detection and Mounting:

  • Incubate with fluorophore-conjugated secondary antibody (e.g., Alexa Fluor 555/647) at 1:500 dilution for 1 hour at room temperature

  • Counter-stain nuclei with DAPI (1 μg/ml) for 5 minutes

  • Mount with anti-fade mounting medium

• Imaging Considerations:

  • Use confocal microscopy for precise nuclear localization

  • Employ z-stack imaging to capture the full nuclear volume

  • Compare with non-phospho LMNA antibody staining to distinguish localization differences

Research has shown that phosphorylated LMNA (S392) localizes predominantly in the nuclear interior, while non-phosphorylated LMNA is found at the nuclear periphery . This distinct localization pattern can serve as an internal validation of staining specificity. For dual staining experiments, researchers should consider using anti-phospho-S22-LMNA as a complementary marker, as S22 and S392 phosphorylation often occur together .

What is the functional significance of LMNA S392 phosphorylation in cell cycle regulation?

LMNA S392 phosphorylation plays a crucial role in cell cycle regulation, particularly during mitosis. Research has demonstrated that:

• S392 phosphorylation, along with S22 phosphorylation, contributes to nuclear lamina disassembly during the G2-to-M transition

• Phosphorylation at these sites is mediated by cyclin-dependent kinases and peaks during mitosis when the nuclear envelope breaks down

• During interphase, a basal level of S392-phosphorylated LMNA exists in the nuclear interior, distinct from the non-phosphorylated form at the nuclear periphery

Cell cycle synchronization experiments have shown that while phosphorylated LMNA peaks during G2/M phase, it remains detectable during G0 and G1/S phases, indicating additional functions beyond mitotic nuclear envelope breakdown . Flow cytometry analysis has confirmed the presence of phosphorylated LMNA in all cell cycle phases, suggesting constitutive roles in nuclear organization throughout interphase . This differential phosphorylation state creates functional subpopulations of LMNA with distinct properties and cellular functions throughout the cell cycle.

How does S392 phosphorylation affect LMNA interactions with chromatin?

S392 phosphorylation significantly alters LMNA interactions with chromatin in several important ways:

• Phosphorylated LMNA (S392) preferentially binds to genomic sites outside of traditional lamina-associated domains (LADs)

• ChIP-seq analyses have shown that phospho-mimetic LMNA (S22D/S392D), particularly in the form of Lamin C, strongly binds to active enhancers in euchromatin regions

• Phospho-mimetic Lamin C (S22D/S392D) displays significantly higher affinity for these genomic sites compared to phospho-mimetic Lamin A

These findings reveal that phosphorylation acts as a molecular switch that redirects LMNA from the nuclear periphery to the nuclear interior, where it can interact with and potentially regulate active chromatin regions . The differential binding patterns between phosphorylated and non-phosphorylated LMNA suggest important roles in gene expression regulation. Statistical analyses have shown that this differential binding is highly significant (P=1×10^-8), highlighting the biological importance of this phosphorylation-dependent chromatin interaction .

What signaling pathways regulate LMNA S392 phosphorylation?

LMNA S392 phosphorylation is regulated by several signaling pathways:

• MAP Kinase Signaling: Oncogenic Ras activates the MAPK pathway, which directly targets LMNA S392 for phosphorylation

• Cell Cycle-Dependent Phosphorylation: Cyclin-dependent kinases, particularly CDK1, phosphorylate LMNA at S392 during the G2/M transition

• Mechanical Stress Response: Phosphorylation at S392, along with S22, has been observed in response to mechanical stress in the cellular microenvironment

These diverse regulatory mechanisms highlight the multifunctional nature of LMNA phosphorylation in cellular processes. The oncogenic Ras-MAPK axis is particularly significant as it connects LMNA phosphorylation to cancer cell signaling networks . Knockdown experiments using shRNAs targeting KRAS have demonstrated altered LMNA phosphorylation patterns, confirming the link between these pathways . Understanding these regulatory mechanisms provides insights into how LMNA phosphorylation may contribute to disease states, particularly in cancer contexts where Ras signaling is frequently dysregulated.

How can researchers distinguish between phosphorylated forms of Lamin A versus Lamin C using the Phospho-LMNA (S392) Antibody?

Distinguishing between phosphorylated Lamin A and Lamin C requires careful experimental design and data interpretation:

• Western Blot Analysis:

  • Lamin A appears at approximately 70 kDa

  • Lamin C appears at approximately 65 kDa

  • Use high-resolution gels (8-10% acrylamide) with extended running time to achieve clear band separation

  • Dual staining with both phospho-specific and pan-LMNA antibodies on sequential blots can confirm isoform identity

• Genetic Approaches:

  • Utilize Lamin A or Lamin C-specific knockout models

  • Express Lamin A-only or Lamin C-only constructs in LMNA-/- cells

• Functional Analysis:

  • Research has shown that phospho-mimetic Lamin C (S22D/S392D) binds more strongly to specific genomic sites than phospho-mimetic Lamin A, with statistical significance (P=1×10^-8)

  • This differential binding can be used to infer which isoform is predominantly contributing to observed effects

When interpreting results, researchers should consider that phosphorylated Lamin C appears to have stronger functional effects in certain contexts, particularly in binding to active enhancers in the nuclear interior . The phosphorylation status and functional differences between these isoforms may contribute to their distinct roles in nuclear organization and gene regulation.

What are common pitfalls in Western blotting with Phospho-LMNA (S392) Antibody and how can they be avoided?

Several common challenges arise when performing Western blotting with Phospho-LMNA (S392) Antibody:

• Loss of Phospho-Epitope:

  • Problem: Phosphatases in cell lysates can dephosphorylate LMNA during sample preparation

  • Solution: Include phosphatase inhibitors (e.g., 1 mM NaVO₃, 10 mM NaF) in lysis buffers

  • Add EDTA (1 mM) to chelate metal ions required for phosphatase activity

• High Background:

  • Problem: Non-specific binding, especially with polyclonal antibodies

  • Solution: Optimize blocking (5% BSA is preferred over milk for phospho-epitopes)

  • Increase washing duration and detergent concentration (0.1-0.2% Tween-20)

  • Use 0.5% BSA in antibody dilution buffer as described in protocols

• Multiple Bands:

  • Problem: Detection of degradation products or cross-reactivity

  • Solution: Confirm band identity with LMNA knockout controls

  • Compare with total LMNA antibody to identify specific bands

  • Use freshly prepared samples and optimize protease inhibitor cocktails

• Variable Signal Intensity:

  • Problem: Phosphorylation levels vary with cell cycle stage and culture conditions

  • Solution: Synchronize cells when comparing experimental conditions

  • Normalize phospho-LMNA signals to total LMNA levels

  • Include positive controls (e.g., mitotic cells or Ras-activated cells)

Researchers should also be aware that the recommended dilution range (1:500-1:2000) may need optimization for specific experimental systems . Preliminary experiments with different antibody concentrations can help identify the optimal conditions for each research application.

How can researchers validate the specificity of Phospho-LMNA (S392) Antibody in their experimental system?

Validating antibody specificity is crucial for reliable research findings. For Phospho-LMNA (S392) Antibody, several approaches are recommended:

• Genetic Validation:

  • Test antibody reactivity in LMNA knockout (LMNA-/-) cells or tissues, which should show no signal

  • Reintroduce wild-type LMNA to rescue the signal

  • Compare with non-phosphorylatable mutant (S392A) expression, which should show no signal with the phospho-specific antibody

• Biochemical Validation:

  • Treat samples with lambda phosphatase to remove phosphorylation and confirm signal reduction

  • Perform peptide competition assays using the phosphorylated peptide immunogen

  • Compare signal with antibodies recognizing total LMNA to confirm specificity for the phosphorylated form

• Physiological Validation:

  • Modulate known upstream regulators (e.g., Ras signaling or CDK inhibitors) and observe expected changes in signal intensity

  • Examine cell cycle-dependent changes in phosphorylation levels

  • Assess subcellular localization patterns, as phosphorylated LMNA (S392) should predominate in the nuclear interior rather than at the nuclear periphery

• Technical Controls:

  • Include secondary antibody-only controls to assess background

  • Use isotype control antibodies to evaluate non-specific binding

  • Perform immunoprecipitation followed by mass spectrometry to confirm the identity of the recognized protein

These validation approaches build confidence in the specificity of the antibody and the biological relevance of observed signals, particularly when multiple validation methods yield consistent results .

How can researchers use Phospho-LMNA (S392) Antibody in ChIP-seq experiments to study chromatin interactions?

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) with Phospho-LMNA (S392) Antibody provides valuable insights into chromatin interactions:

• Experimental Design:

  • Optimize crosslinking conditions (1% formaldehyde for 10 minutes is typical)

  • Use sonication to achieve chromatin fragments of 200-500 bp

  • Incubate chromatin with Phospho-LMNA (S392) Antibody (4-10 μg per ChIP reaction)

  • Include input controls and LMNA knockout negative controls

  • Consider parallel ChIP with non-phospho LMNA antibody for comparative analysis

• Data Analysis Approach:

  • Compare binding profiles of phosphorylated versus non-phosphorylated LMNA

  • Analyze enrichment at enhancers, promoters, and lamina-associated domains

  • Integrate with RNA-seq and histone modification ChIP-seq data

• Key Findings from Literature:

  • Phosphorylated LMNA (particularly phospho-mimetic S22D/S392D Lamin C) shows strong binding to genomic sites outside traditional lamina-associated domains

  • These binding sites often correspond to active enhancers in euchromatin regions

  • Statistical analysis shows significant enrichment compared to non-phosphorylated forms (P=1×10^-8)

This approach has revealed that phosphorylation status dramatically alters the genomic binding profile of LMNA, suggesting a mechanism for how post-translational modifications of nuclear lamina proteins can influence gene expression . Researchers should be aware that ChIP-seq with phospho-specific antibodies may require additional optimization steps compared to standard ChIP protocols to preserve the phosphorylation state throughout the procedure.

What are the implications of LMNA S392 phosphorylation in cancer research and potential therapeutic approaches?

LMNA S392 phosphorylation has significant implications for cancer research:

• Oncogenic Signaling:

  • S392 has been identified as a specific target of oncogenic Ras-mediated MAP kinase signaling

  • This connects LMNA phosphorylation to a major oncogenic pathway active in many human cancers

• Gene Regulation:

  • Phosphorylated LMNA binds to active enhancers in the nuclear interior, potentially regulating oncogene expression

  • Altered LMNA phosphorylation may contribute to dysregulated gene expression in cancer cells

• Therapeutic Potential:

  • Inhibiting kinases responsible for LMNA S392 phosphorylation could disrupt oncogenic gene expression programs

  • Developing compounds that specifically block the interaction between phosphorylated LMNA and chromatin could represent a novel therapeutic approach

  • Using phosphorylated LMNA as a biomarker might help identify tumors with hyperactive Ras-MAPK signaling

• Experimental Models:

  • KRAS knockdown experiments have demonstrated altered phosphorylation patterns of target proteins including LMNA

  • Cell lines with different KRAS mutation status could be used to study LMNA phosphorylation dynamics

These findings suggest that LMNA phosphorylation could be both a consequence and a mediator of oncogenic signaling, positioning it as a potential therapeutic target or diagnostic marker in cancer . Further research is needed to fully elucidate how LMNA phosphorylation contributes to the cancer phenotype and whether targeting this modification could have clinical benefits.

How does LMNA S392 phosphorylation interact with other post-translational modifications of LMNA?

LMNA undergoes multiple post-translational modifications that interact in complex ways:

• Coordinated Phosphorylation:

  • S392 phosphorylation often occurs in coordination with S22 phosphorylation

  • Phospho-mimetic experiments with double mutants (S22D/S392D) show stronger effects than single phosphorylation site mutations

  • This suggests cooperative effects between these phosphorylation sites

• Regulatory Interplay:

  • Phosphorylation at S392 may influence other modifications such as:

    • Farnesylation of the C-terminal CaaX motif

    • Sumoylation at multiple lysine residues

    • Acetylation of lysine residues

  • These modifications collectively determine LMNA localization, interaction partners, and stability

• Functional Consequences:

  • Multiple phosphorylation events trigger conformational changes that expose or mask interaction domains

  • Different combinations of modifications likely create functionally distinct subpopulations of LMNA within the cell

  • Mass spectrometry approaches have identified modification "signatures" that correlate with specific cellular states

• Technical Approaches:

  • Use phospho-specific antibodies in combination with antibodies recognizing other modifications

  • Employ mass spectrometry-based proteomics to map modification patterns

  • Create combinatorial mutation constructs to study modification interdependencies