Phospho-MAP4 (S696) Antibody

What is Phospho-MAP4 (S696) Antibody and what does it detect?

Phospho-MAP4 (S696) antibody specifically recognizes microtubule-associated protein 4 (MAP4) only when phosphorylated at the Serine 696 position. This antibody detects endogenous levels of MAP4 protein in its phosphorylated state, making it valuable for studying post-translational modifications that regulate MAP4 function. The antibody is typically generated using synthetic peptides derived from human MAP4 surrounding the phosphorylation site of Ser696 (amino acids 662-711) .

What species does Phospho-MAP4 (S696) Antibody react with?

Most commercially available Phospho-MAP4 (S696) antibodies demonstrate reactivity across multiple species including:

• Human

• Mouse

• Rat

This cross-reactivity is due to the high conservation of the phosphorylation site and surrounding amino acid sequences across these species .

What applications is the Phospho-MAP4 (S696) Antibody validated for?

Phospho-MAP4 (S696) antibodies have been validated for several experimental applications:

Some antibodies may also be validated for additional applications, but these three represent the most consistently validated uses across different manufacturers .

How should Phospho-MAP4 (S696) Antibody be stored to maintain optimal activity?

For long-term storage of Phospho-MAP4 (S696) antibody:

• Store at -20°C for up to one year in aliquots to avoid repeated freeze-thaw cycles

• For short-term storage (up to one month), keep at 4°C

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

Each freeze-thaw cycle can reduce antibody performance, so it's recommended to make small aliquots when receiving the antibody .

What controls should be included when using Phospho-MAP4 (S696) Antibody?

A robust experimental design using Phospho-MAP4 (S696) antibody should include these controls:

• Positive control: Samples from hypoxia-treated cells or tissues, which induce robust MAP4 phosphorylation at S696

• Negative control:

  • Samples treated with phosphatase to remove phosphate groups

  • Use of blocking peptide (non-phosphorylated version of the immunogen)

• Validation controls:

  • MAP4 knockdown/knockout samples

  • Samples expressing MAP4(Ala) mutant, which mimics the dephosphorylated form

  • Samples expressing MAP4(Glu) mutant, which mimics the phosphorylated form

These controls help confirm specificity and rule out non-specific binding .

What experimental conditions can induce MAP4 phosphorylation at S696 for positive control generation?

To generate positive controls with high levels of MAP4 phosphorylation at S696:

• Hypoxia treatment: Subject cells (particularly cardiomyocytes) to 1% O₂ for 6-12 hours, which robustly induces phosphorylation at S696, S768, and S787 sites

• p38/MAPK pathway activation: Treatment with LPS or TNF-α activates p38/MAPK signaling, leading to MAP4 phosphorylation

• MKK6(Glu) expression: Transfection with constitutively active MKK6(Glu) activates downstream p38/MAPK, resulting in MAP4 phosphorylation

These methods reliably increase phosphorylation at the S696 site, providing effective positive controls for antibody validation experiments .

How can I differentiate between cytosolic and mitochondrial localization of phosphorylated MAP4?

To investigate the subcellular localization of phosphorylated MAP4:

• Subcellular fractionation:

  • Separate mitochondrial, cytosolic, and cytoskeletal fractions using differential centrifugation

  • Validate fraction purity using markers (e.g., VDAC for mitochondria, tubulin for cytoskeleton)

  • Analyze each fraction by Western blot using Phospho-MAP4 (S696) antibody

• Confocal microscopy:

  • Co-stain cells with Phospho-MAP4 (S696) antibody and mitochondrial markers (e.g., MitoTracker)

  • Analyze co-localization using quantitative image analysis

  • Calculate Pearson's correlation coefficient to measure the degree of co-localization

What are the key considerations when using Phospho-MAP4 (S696) Antibody in immunofluorescence studies?

For optimal immunofluorescence results with Phospho-MAP4 (S696) antibody:

• Fixation method: 4% paraformaldehyde fixation for 15 minutes at room temperature preserves phospho-epitopes better than methanol fixation

• Permeabilization: Gentle permeabilization with 0.1% Triton X-100 for 5 minutes maintains antibody accessibility while preserving cellular structures

• Blocking: Use 5% BSA in PBS for 1 hour to reduce non-specific binding

• Antibody dilution: Begin with 1:500 dilution and optimize based on signal-to-noise ratio

• Incubation time: Overnight incubation at 4°C often yields better results than shorter incubations

• Phosphatase inhibitors: Include sodium orthovanadate (1mM) and sodium fluoride (10mM) in all buffers to preserve phosphorylation

When studying mitochondrial translocation, co-staining with mitochondrial markers is essential for accurate localization assessment .

How does MAP4 phosphorylation at S696 affect its interaction with microtubules and what techniques can measure this?

MAP4 phosphorylation at S696 (along with S768 and S787) significantly decreases its binding affinity for microtubules, leading to microtubule destabilization. To study this relationship:

• Co-sedimentation assays:

  • Incubate purified tubulin with either phosphorylated or non-phosphorylated MAP4

  • Centrifuge to pellet polymerized microtubules

  • Analyze pellet and supernatant fractions by Western blot to quantify MAP4 binding

• TIRF microscopy:

  • Label tubulin with fluorescent dyes

  • Add phosphorylated or non-phosphorylated MAP4

  • Monitor microtubule dynamics in real-time

  • Measure growth rates, catastrophe frequency, and rescue events

• MAP4 mutant studies:

  • Express MAP4(Ala) (non-phosphorylatable) or MAP4(Glu) (phosphomimetic) mutants

  • Compare their localization and microtubule-binding properties

  • Assess microtubule stability using techniques like nocodazole resistance assays

Research shows that phosphorylated MAP4 detaches from microtubules and translocates to mitochondria, potentially inducing apoptosis independent of its effects on microtubule dynamics .

What mechanisms connect MAP4 phosphorylation to mitochondrial dysfunction and apoptosis?

The phosphorylation-dependent mitochondrial translocation of MAP4 initiates a cascade of events leading to apoptosis:

• mPTP opening: Phosphorylated MAP4 that translocates to mitochondria promotes mitochondrial permeability transition pore (mPTP) opening

• Mitochondrial membrane potential disruption: This leads to loss of mitochondrial membrane potential (ΔΨm)

• Cytochrome c release: mPTP opening facilitates the release of pro-apoptotic factors like cytochrome c

• Caspase activation: Released cytochrome c activates downstream caspases, initiating apoptosis

Experimental techniques to study this cascade include:

• JC-1 staining to measure mitochondrial membrane potential

• Calcein-AM/Co²⁺ quenching assay to detect mPTP opening

• Immunofluorescence to track cytochrome c release

• Caspase activity assays to measure apoptotic progression

Interestingly, this pro-apoptotic function of phosphorylated MAP4 occurs independently of its effects on microtubule dynamics .

How does the p38/MAPK signaling pathway regulate MAP4 phosphorylation at S696, and how can this be experimentally manipulated?

The p38/MAPK pathway is a key regulator of MAP4 phosphorylation at S696:

• Activation mechanism:

  • Inflammatory stimuli (LPS, TNF-α) activate the p38/MAPK pathway

  • Activated p38 directly or indirectly phosphorylates MAP4 at S696

  • This phosphorylation triggers MAP4 detachment from microtubules and mitochondrial translocation

• Experimental manipulation:

  • Activation: Use MKK6(Glu), a constitutively active upstream kinase, to activate p38/MAPK

  • Inhibition: Apply SB203580 (p38 inhibitor) to block MAP4 phosphorylation

  • Genetic approaches: Express dominant-negative p38 or siRNA knockdown

• Readouts:

  • Western blotting with Phospho-MAP4 (S696) antibody to measure phosphorylation levels

  • Microtubule stability assays to assess downstream effects

  • Endothelial permeability measurements to evaluate functional consequences

Studies show that p38/MAPK-mediated MAP4 phosphorylation contributes to endothelial barrier dysfunction in acute lung injury models and hypoxia-induced cardiomyocyte apoptosis .

How does MAP4 phosphorylation impact cardiac microvascular density and what methods can quantify this effect?

MAP4 phosphorylation has been shown to reduce cardiac microvascular density through several mechanisms:

• Mechanistic pathways:

  • Activation of NLRP3-related pyroptosis in endothelial cells

  • Inhibition of VEGF/VEGFR2 and ANG2/TIE2 angiogenic signaling pathways

  • Induction of endothelial cell apoptosis and mitochondrial disruption

• Quantification methods:

  • Immunofluorescence staining: Using CD31 or CD34 antibodies to visualize and quantify microvasculature

  • Lectin staining: To directly visualize functional blood vessels

  • Protein expression analysis: Measuring levels of angiogenic factors (VEGFA, VEGFR2, ANG2, TIE2)

  • Angiogenesis assays: Tube formation assays with endothelial cells expressing MAP4 mutants

• Experimental models:

  • MAP4 knock-in (KI) mice showing elevated MAP4 phosphorylation

  • Adenoviral expression of MAP4(Glu) to mimic phosphorylated MAP4 in endothelial cells

This research area has significant implications for understanding cardiac remodeling in both young and aged subjects .

What are common issues when using Phospho-MAP4 (S696) Antibody and how can they be resolved?

When working with Phospho-MAP4 (S696) antibody, researchers may encounter several common problems:

• High background in immunostaining:

  • Cause: Insufficient blocking or non-specific antibody binding

  • Solution: Increase blocking time/concentration, optimize antibody dilution, include 0.1% Tween-20 in wash buffers

• Weak or no signal in Western blot:

  • Cause: Low phosphorylation levels or phosphatase activity during sample preparation

  • Solution: Include phosphatase inhibitors (sodium orthovanadate, sodium fluoride) in lysis buffers, use positive controls like hypoxia-treated samples

• Multiple bands in Western blot:

  • Cause: MAP4 has multiple isoforms (ranging from 70-121 kDa) and can be differentially phosphorylated

  • Solution: Verify band specificity using phosphatase treatment or MAP4 knockdown controls

• Inconsistent results between experiments:

  • Cause: Variable phosphorylation status due to cell culture conditions

  • Solution: Standardize cell culture conditions, serum starvation before treatments, synchronize cells .

How can I distinguish between specific and non-specific signals when using Phospho-MAP4 (S696) Antibody?

To distinguish between specific and non-specific signals:

• Validation experiments:

  • Peptide competition: Pre-incubate antibody with phosphorylated immunogen peptide (should block specific signal)

  • Lambda phosphatase treatment: Treat one sample set with phosphatase to remove phosphorylation (should eliminate specific signal)

  • siRNA knockdown: Compare signal in MAP4 knockdown versus control cells

• Control antibodies:

  • Use total MAP4 antibody in parallel to confirm protein presence

  • Compare phospho-specific signal pattern with total protein pattern

• Signal characteristics:

  • Specific phospho-MAP4 (S696) signal should increase with treatments known to induce phosphorylation (hypoxia, p38/MAPK activation)

  • Signal should localize appropriately (cytoskeletal in normal conditions, shifting to mitochondrial under stress)

  • Primary band should appear at the expected molecular weight (~121 kDa) .

How do I interpret conflicting results between phosphorylation status and functional outcomes in MAP4 studies?

When facing conflicting results in phosphorylated MAP4 studies:

• Consider multiple phosphorylation sites:

  • MAP4 has multiple phosphorylation sites (S696, S768, S787 in humans) that may have different or cooperative effects

  • Use antibodies specific to different phosphorylation sites to create a complete phosphorylation profile

• Examine temporal dynamics:

  • MAP4 phosphorylation is dynamic and timing matters

  • Create detailed time courses to capture transient effects

  • Different downstream effects may occur at different time points after phosphorylation

• Cell type specificity:

  • MAP4 functions may vary between cell types (cardiomyocytes vs. endothelial cells)

  • Different cellular contexts may have distinct signaling networks that modify MAP4 function

• Consider compensatory mechanisms:

  • Cells may activate compensatory pathways when MAP4 is phosphorylated

  • Use systems biology approaches to capture network-level responses

• Validate with multiple approaches:

  • Combine genetic approaches (MAP4 mutants) with pharmacological ones

  • Use both gain-of-function and loss-of-function experiments .

What are the latest research developments involving MAP4 phosphorylation at S696 in cardiovascular diseases?

Recent research has revealed several important roles of MAP4 phosphorylation in cardiovascular pathology:

• Cardiac remodeling:

  • Phosphorylated MAP4 contributes to cardiac remodeling in an age-dependent manner

  • MAP4 knock-in mice with elevated phosphorylation levels show progressive cardiac dysfunction

• Microvascular density reduction:

  • Phosphorylated MAP4 decreases cardiac microvascular density by:

    • Inhibiting VEGF/VEGFR2 and ANG2/TIE2 angiogenic pathways

    • Activating NLRP3-related pyroptosis in endothelial cells

    • Inducing mitochondrial dysfunction and apoptosis in cardiac endothelial cells

• Endothelial barrier dysfunction:

  • p38/MAPK-mediated MAP4 phosphorylation destabilizes microtubules in endothelial cells

  • This leads to increased vascular permeability and edema formation

  • May contribute to acute lung injury and acute respiratory distress syndrome

• Therapeutic targeting:

  • Inhibition of MAP4 phosphorylation (using MAP4(Ala) mutants) suppresses mitochondrial translocation and apoptosis

  • NLRP3 inflammasome blockade alleviates the inhibited angiogenic ability induced by phosphorylated MAP4

These findings suggest MAP4 phosphorylation as a potential therapeutic target for cardiac remodeling and vascular dysfunction .

How can advanced microscopy techniques enhance our understanding of phosphorylated MAP4 dynamics and function?

Advanced microscopy approaches offer powerful new ways to study phosphorylated MAP4:

• Live-cell imaging with phospho-specific sensors:

  • Genetically encoded FRET-based sensors to monitor MAP4 phosphorylation in real-time

  • Allows observation of spatial and temporal dynamics of phosphorylation events

  • Can reveal localized phosphorylation within specific cellular compartments

• Super-resolution microscopy:

  • STORM or PALM imaging to visualize MAP4-microtubule interactions at nanometer resolution

  • Can detect changes in microtubule architecture upon MAP4 phosphorylation

  • Enables precise localization of phosphorylated MAP4 within mitochondria

• Correlative light and electron microscopy (CLEM):

  • Combines fluorescence localization of phosphorylated MAP4 with ultrastructural context

  • Particularly valuable for studying mitochondrial morphology changes induced by p-MAP4

• Intravital microscopy:

  • Monitor vascular permeability changes in animal models with altered MAP4 phosphorylation

  • Track endothelial cell responses in real-time in vivo

  • Can be combined with genetic reporters to simultaneously monitor multiple parameters

These advanced techniques can help resolve conflicting data by providing spatial, temporal, and contextual information about MAP4 phosphorylation events .

What are the most reliable antibody validation resources for Phospho-MAP4 (S696) Antibody?

When selecting and validating Phospho-MAP4 (S696) antibody, consider these resources:

• Primary literature validation:

  • Studies by Hu et al. (2014) in Cell Death & Disease demonstrating specificity for phosphorylated MAP4 in cardiac tissues

  • Research by Li et al. (2021) showing application in cardiac microvascular studies

  • Publications using phospho-site specific mutants (MAP4-Ala or MAP4-Glu) as controls

• Database resources:

  • Antibodypedia database entries for independent validation

  • International Working Group for Antibody Validation (IWGAV) guidelines

  • CiteAb citation database to identify highly-cited antibody products

• Validation methodology:

  • Look for antibodies validated with multiple approaches (Western blot, IF, IHC)

  • Seek evidence of knockout/knockdown controls

  • Check for phosphatase treatment validation

  • Verify cross-reactivity testing across species

• Commercial validation data:

  • Manufacturer datasheets showing clear single-band Western blots

  • Side-by-side comparisons with total MAP4 antibodies

  • Validation in relevant disease models (hypoxia, ischemia) .