Phospho-VIM (Ser56) Antibody

What is Phospho-Vimentin (Ser56) and what biological processes is it involved in?

Phospho-Vimentin (Ser56) represents vimentin protein phosphorylated at serine position 56. Vimentin is a type III intermediate filament protein found predominantly in mesenchymal cells and forms part of the cytoskeleton alongside microtubules and actin microfilaments . Phosphorylation at Ser56 plays critical roles in several biological processes:

• Neutrophil secretion: Phosphorylation of vimentin at Ser56 is linked to GTP-induced secretion by neutrophils during inflammatory responses

• Mitotic regulation: CDK1 phosphorylates vimentin at Ser56 during mitosis, which provides a binding site for PLK (Polo-like kinase) interaction

• Cellular migration: Altered phosphorylation status at Ser56 affects cytoskeletal dynamics related to cell motility

• Cancer cell properties: Stabilizing vimentin phosphorylation at Ser56 inhibits stem-like cell properties in certain cancer contexts

• Extracellular matrix organization: Phosphorylation changes at vimentin Ser56 can influence fibronectin matrix organization

What applications are Phospho-Vimentin (Ser56) antibodies most commonly used for?

Phospho-Vimentin (Ser56) antibodies are utilized across multiple experimental applications, with each providing unique insights:

• Western Blot (WB): Most commonly used application with dilution ranges typically between 1:500-1:2000. This technique allows visualization of phosphorylated vimentin at approximately 57 kDa

• Immunohistochemistry (IHC): Used to visualize the cellular and tissue localization of phosphorylated vimentin, with recommended dilutions around 1:100-1:300

• Immunofluorescence (IF): Particularly valuable for subcellular localization studies and co-localization with other proteins, typically using dilutions of 1:50-1:200

• ELISA: Used for quantitative detection of phosphorylated vimentin, often with higher dilutions (1:10000-1:20000)

For optimal results, experimental conditions should be determined empirically for each specific application.

How should I select between polyclonal and monoclonal Phospho-Vimentin (Ser56) antibodies?

Selection between polyclonal and monoclonal antibodies should be based on specific experimental requirements:

Polyclonal Phospho-Vimentin (Ser56) antibodies:

• Advantages: Recognize multiple epitopes around the phosphorylation site, potentially offering enhanced sensitivity and robustness to minor changes in protein conformation

• Applications: Well-suited for detection of low-abundance phosphorylated proteins

• Example products: Rabbit polyclonal antibodies are most common

Monoclonal Phospho-Vimentin (Ser56) antibodies:

• Advantages: Highly specific for a single epitope, offering excellent reproducibility between experiments

• Applications: Ideal for applications requiring consistent results across multiple experiments

• Example products: Mouse monoclonal antibodies such as the GT11512 clone

Consider factors such as experimental reproducibility requirements, target abundance, and specific application needs when selecting between these antibody types.

What kinases phosphorylate vimentin at Ser56 in different cellular contexts, and how can I differentiate their activities?

Multiple kinases can phosphorylate vimentin at Ser56 in a context-dependent manner:

• Cyclin-dependent kinase 5 (Cdk5):

  • Context: Mediates vimentin Ser56 phosphorylation during GTP-induced neutrophil secretion

  • Verification method: Use specific Cdk5 inhibitors (roscovitine, olomoucine) or Cdk5 siRNA transfection

  • Finding: Inhibition of Cdk5 significantly reduces GTP-induced (but not fMLP-induced) vimentin Ser56 phosphorylation

• Cyclin-dependent kinase 1 (CDK1):

  • Context: Phosphorylates vimentin at Ser56 during mitosis

  • Verification method: Cell cycle synchronization followed by mitotic arrest with specific agents

  • Finding: This phosphorylation provides a binding site for Polo-like kinase (PLK) interaction

• p21-activated kinase (PAK):

  • Context: Potentially responsible for early phosphorylation of vimentin Ser56 in neutrophils

  • Experimental approach: PAK-specific inhibitors or genetic manipulation

To differentiate between these kinases:

• Use specific inhibitors in combination with time-course studies

• Apply genetic approaches (siRNA, CRISPR-Cas9)

• Conduct kinase assays with recombinant proteins

• Employ phospho-proteomic approaches with kinase-specific contexts

For example, research has shown that in neutrophils stimulated with GTP, initial phosphorylation of vimentin at Ser56 occurs independently of Cdk5, while sustained phosphorylation (5-15 minutes post-stimulation) is predominantly Cdk5-dependent .

How does phosphorylation status at vimentin Ser56 influence cancer stem cell properties and how can it be manipulated experimentally?

Phosphorylation at vimentin Ser56 critically influences cancer stem cell (CSC) properties, particularly in breast cancer models:

Experimental findings on vimentin Ser56 phosphorylation and cancer stemness:

• Stabilizing vimentin phosphorylation at Ser56 using the compound FiVe1 or expression of phosphomimetic VIM-S56E (serine to glutamic acid) mutation leads to:

  • Increased multinucleation specifically in vimentin-expressing hybrid Epithelial/Mesenchymal (E/M) cells

  • Significant inhibition of stemness as measured by mammosphere formation assays

  • Greater effects in cancer stem cell-enriched populations

• Expression of phospho-ablative VIM-S56A (serine to alanine) mutation also induces multinucleation, suggesting that normal cycling of phosphorylation at this site is critical for maintaining CSC properties

Experimental manipulation approaches:

• Pharmacological approach: Use FiVe1 compound to stabilize vimentin Ser56 phosphorylation

• Genetic approaches:

  • Create doxycycline-inducible cell lines expressing phosphomimetic (S56E) vimentin

  • Create doxycycline-inducible cell lines expressing phospho-ablative (S56A) vimentin

• Cell population enrichment:

  • Grow cells in serum-free suspension culture to enrich for CSCs before manipulation

  • Compare with non-enriched monolayer cultures

Critical experimental finding: Stemness properties appear necessary for vimentin-associated multinucleation, as epithelial cells lacking stemness properties (MCF7 and MDA-MB-453) do not exhibit multinucleation even after expression of phosphomimetic VIM-S56E .

What experimental considerations are critical when using Phospho-Vimentin (Ser56) antibodies in co-localization studies?

When conducting co-localization studies with Phospho-Vimentin (Ser56) antibodies, several critical experimental factors must be considered:

Technical considerations:

• Fixation method: Phospho-epitopes are sensitive to fixation conditions. Use fresh paraformaldehyde (4%) and avoid prolonged fixation that may mask phospho-epitopes

• Permeabilization protocol: Use gentle detergents (0.1-0.2% Triton X-100) to preserve phosphorylation status

• Blocking solutions: Ensure blocking agents do not contain phosphatases that could dephosphorylate your target

• Antibody validation: Confirm specificity using phosphatase treatment controls

• Signal-to-noise optimization: Use appropriate dilutions (typically 1:50-1:200 for IF) to maximize signal while minimizing background

Biological considerations:

• Temporal dynamics: Phosphorylation at Ser56 shows distinct temporal patterns. For example, in GTP-stimulated neutrophils, initial phosphorylation occurs at 1-3 minutes, with significant increases at 5-15 minutes

• Cellular compartmentalization: Phospho-vimentin Ser56 distribution changes over time. In neutrophils, co-localization with Cdk5 increases over time following stimulation

• Cell cycle stage: Consider synchronizing cells when studying mitosis-related phosphorylation events

• Stimulation conditions: Different stimuli (e.g., GTP vs. fMLP in neutrophils) induce phosphorylation via different kinases and with different localization patterns

Example protocol from literature:
In neutrophil studies examining Cdk5 and phospho-vimentin Ser56 co-localization:

• Fixation: 4% paraformaldehyde for 15 minutes

• Permeabilization: 0.1% Triton X-100 for 5 minutes

• Primary antibody incubation: Anti-Cdk5 and phospho-vimentin Ser56 antibodies (overnight at 4°C)

• Visualization: Fluorophore-conjugated secondary antibodies

• Advanced imaging: Deconvolution microscopy to clearly visualize partial co-localization

How does the phosphorylation status of vimentin Ser56 influence intermediate filament dynamics and cellular functions?

The phosphorylation status of vimentin Ser56 critically regulates intermediate filament assembly/disassembly dynamics and subsequent cellular functions:

Filament assembly/disassembly dynamics:

• Phosphorylation at Ser56 promotes filament disassembly, particularly during mitosis

• This phosphorylation site is one of several that regulate the dynamic reorganization of the vimentin network

• The cycling between phosphorylated and dephosphorylated states appears critical, as both phosphomimetic (S56E) and phospho-ablative (S56A) mutations disrupt normal filament dynamics

Functional consequences by cell type:

• Neutrophils:

  • GTP-induced phosphorylation of vimentin at Ser56 mediates secretion from primary, secondary, and tertiary granules

  • The secretory process shows time-dependent correlation with vimentin Ser56 phosphorylation

  • Antibody-mediated inhibition of vimentin Ser56 phosphorylation reduces GTP-induced secretion

• Cancer cells:

  • Disruption of normal vimentin Ser56 phosphorylation cycling (via either S56E or S56A mutations) leads to multinucleation specifically in cells with stem-like properties

  • This disruption inhibits mammosphere formation, indicating interference with cancer stem cell self-renewal capacity

• Endothelial cells:

  • Phosphorylation at vimentin Ser39 (not Ser56) appears more critical for fibronectin matrix organization in response to Treponema pallidum infection

Experimental approaches to study these dynamics:

• Time-course immunofluorescence to track phosphorylation changes and filament reorganization

• Live-cell imaging with phospho-specific antibodies or phosphomimetic/phospho-ablative mutants

• Correlative functional assays (e.g., secretion, migration, division) coupled with phosphorylation status monitoring

What controls should be implemented when using Phospho-Vimentin (Ser56) antibodies in experimental settings?

Implementing appropriate controls is critical for ensuring reliable and interpretable results when using Phospho-Vimentin (Ser56) antibodies:

Essential experimental controls:

• Specificity controls:

  • Phosphatase treatment: Samples treated with lambda phosphatase should show reduced or eliminated signal

  • Phospho-blocking peptide: Pre-incubation of antibody with the phosphorylated peptide used as immunogen should eliminate specific signal

  • Non-phosphorylated peptide competition: Pre-incubation with non-phosphorylated peptide should not affect specific signal

• Biological controls:

  • Positive control samples: Use cells or tissues with known high levels of vimentin Ser56 phosphorylation

    • Example: HeLa cells treated with paclitaxel (as mentioned in product information)

    • Example: Neutrophils stimulated with GTP for 5-15 minutes

  • Negative control samples: Use cells with vimentin knockout or cells where phosphorylation is inhibited

    • Example: Cells treated with appropriate kinase inhibitors (roscovitine for Cdk5-mediated phosphorylation)

• Technical controls:

  • Isotype control: Use matched isotype IgG at the same concentration to assess non-specific binding

  • Secondary antibody only: Control for secondary antibody background signal

  • Loading controls: For western blots, include total vimentin detection and housekeeping proteins

  • Cross-reactivity assessments: Test against similar phosphorylation sites (e.g., other serine phosphorylation sites on vimentin)

• Validation approaches:

  • siRNA knockdown: Reduction of total vimentin should correlate with reduction in phospho-signal

  • Genetic approaches: Use of phospho-mimetic (S56E) or phospho-ablative (S56A) vimentin mutants

  • Multiple detection methods: Confirm results using different techniques (e.g., IF and WB)

  • Multiple antibody sources: When possible, confirm key findings with antibodies from different manufacturers

Protocol example for phosphatase control:

• Split your sample into two aliquots

• Treat one aliquot with lambda phosphatase (400-800 units) for 30 minutes at 30°C

• Process both samples identically for western blot or immunostaining

• Compare signal intensity between treated and untreated samples

How do different stimuli influence the kinetics and localization of vimentin Ser56 phosphorylation?

Different stimuli induce distinct patterns of vimentin Ser56 phosphorylation with varying kinetics, localization, and kinase dependencies:

GTP-induced phosphorylation in neutrophils:

• Early phase (1-3 minutes):

  • Initial phosphorylation occurs independently of Cdk5

  • Limited co-localization with Cdk5

  • Potentially mediated by PAK or other kinases

• Later phase (5-15 minutes):

  • Significantly increased phosphorylation predominantly dependent on Cdk5

  • Increased co-localization with Cdk5 in cytoplasmic compartments

  • Strong correlation with granule secretion

• Inhibition profile:

  • Roscovitine and olomoucine (Cdk5 inhibitors) significantly reduce phosphorylation

  • Cdk5 siRNA similarly reduces phosphorylation

  • Incomplete inhibition suggests involvement of additional kinases

fMLP-induced phosphorylation in neutrophils:

• Temporal pattern: Progressive increase in phosphorylation over time

• Kinase dependency: Not dependent on Cdk5 (roscovitine does not inhibit phosphorylation)

• Downstream effects: Despite Cdk5-independent phosphorylation, Cdk5 still regulates secretion through separate mechanisms

• Potential mediators: Possibly related to cGMP signaling

Mitotic phosphorylation:

• Timing: Occurs during specific phases of mitosis

• Kinase responsible: CDK1 (not Cdk5)

• Consequence: Provides binding site for PLK, leading to subsequent phosphorylation at Ser82/83

Experimental approach to study stimulus-specific phosphorylation:

• Time-course analysis: Collect samples at multiple timepoints (1, 3, 5, 10, 15 minutes) following stimulation

• Co-localization studies: Use confocal or deconvolution microscopy to examine spatial relationships with relevant kinases

• Pharmacological inhibitors: Apply kinase-specific inhibitors to identify responsible enzymes

• Correlation with functional outcomes: Measure related cellular functions (e.g., secretion, migration) at the same timepoints

What experimental strategies can resolve contradictory findings regarding vimentin Ser56 phosphorylation in different cellular contexts?

When faced with contradictory findings regarding vimentin Ser56 phosphorylation across different research contexts, several experimental strategies can help resolve these discrepancies:

Context-specific kinase identification:

• Approach: Use pharmacological inhibitors, genetic knockdown, and kinase assays to identify the specific kinases responsible in each context

• Example: In neutrophils, Cdk5 mediates GTP-induced but not fMLP-induced vimentin Ser56 phosphorylation, highlighting stimulus-specific mechanisms

• Implementation:

  • Panel of specific kinase inhibitors

  • siRNA/shRNA knockdown

  • Kinase-dead dominant negative constructs

  • In vitro kinase assays with recombinant proteins

Temporal resolution studies:

• Approach: High-resolution time-course studies to capture rapid phosphorylation dynamics

• Example: In GTP-stimulated neutrophils, initial phosphorylation (1-3 min) occurs independently of Cdk5, while later phosphorylation (5-15 min) is Cdk5-dependent

• Implementation:

  • Multiple short-interval timepoints

  • Live-cell imaging with phospho-specific sensors

  • Rapid cell lysis techniques to preserve phosphorylation status

Subcellular compartment analysis:

• Approach: Isolate and analyze distinct subcellular fractions to detect compartment-specific phosphorylation

• Example: Vimentin pSer56 and Cdk5 colocalization increases in specific cytoplasmic compartments over time after GTP stimulation

• Implementation:

  • Subcellular fractionation followed by western blot

  • Super-resolution microscopy

  • Proximity ligation assays

Validation across multiple cell types:

• Approach: Compare phosphorylation patterns in different cell types with the same stimulus

• Example: Vimentin phosphorylation responds differently in hybrid E/M cancer cells versus purely mesenchymal cells

• Implementation:

  • Panel of cell lines representing different lineages

  • Primary cells compared to immortalized lines

  • Cells at different stages of differentiation

Analysis of concurrent modifications:

• Approach: Examine multiple post-translational modifications simultaneously

• Rationale: Phosphorylation at one site may influence or be influenced by modifications at other sites

• Implementation:

  • Multi-plex phospho-antibody arrays

  • Mass spectrometry-based phospho-proteomics

  • Sequential immunoprecipitation

Functional correlation studies:

• Approach: Correlate phosphorylation status with specific cellular outcomes

• Example: In neutrophils, vimentin Ser56 phosphorylation correlates with granule secretion for GTP but not fMLP stimulation

• Implementation:

  • Side-by-side analysis of phosphorylation and functional readouts

  • Phosphomimetic and phospho-ablative mutants to confirm functional links

  • Antibody-mediated inhibition of phosphorylation

By systematically applying these strategies, researchers can identify context-specific mechanisms and resolve apparently contradictory findings regarding vimentin Ser56 phosphorylation across different experimental systems.

Scientific Research Highlight: Vimentin Ser56 Phosphorylation in Cell Model Systems

Vimentin Ser56 Phosphorylation Patterns in Different Cellular Contexts