CEP4 Antibody

What is CEP4 and what cellular functions does it regulate?

CEP4 (Cdc42 effector protein-4) is a substrate of multiple PKC isoforms in human cells that plays a significant role in cell motility and cytoskeletal organization. As a Cdc42-binding protein, CEP4 participates in signaling pathways that regulate actin dynamics. Research shows that CEP4 can undergo phosphorylation which dramatically alters its binding partners and cellular functions .

What are the recognized phosphorylation sites on CEP4 and their significance?

CEP4 contains multiple serine residues that can potentially be phosphorylated, but research has identified two critical phosphorylation sites that significantly alter CEP4 function: Ser18 and Ser80. These sites are embedded in PKC consensus sequences and have been verified by mass spectrometry (MS/MS) analysis as direct targets of PKCα in vitro .

The phosphorylation status of these sites dramatically affects CEP4's binding partners and cellular functions:

The combined phosphorylation at both sites produces a stronger effect than either site alone, suggesting synergistic functionality in regulating CEP4's role in cell motility .

How does CEP4 differ from other CEP family members?

CEP4 stands out among the five CEP isoforms due to its unique regulatory mechanism. Unlike the other four CEP isoforms, only CEP4 possesses PKC phosphorylation sites, making it exclusively responsive to regulation by PKC . This distinctive characteristic suggests that CEP4 has evolved specialized functions in PKC-mediated signaling pathways.

When comparing CEP isoforms:

• CEP4 contains PKC phosphorylation sites (Ser18 and Ser80)

• Other CEP isoforms (CEP1, CEP2, CEP3, and CEP5) lack these PKC phosphorylation sites

• CEP4 can dissociate from Cdc42 upon phosphorylation

• CEP4 uniquely forms complexes with proteins like TEM4 when phosphorylated

This unique regulatory capacity of CEP4 suggests it plays specialized roles in cellular processes, particularly those involving PKC signaling, that are distinct from other members of the CEP family .

What criteria should researchers use when selecting CEP4 antibodies for specific applications?

When selecting CEP4 antibodies for research applications, consider these critical criteria to ensure experimental success:

• Phosphorylation-state specificity: For studies investigating CEP4 phosphorylation, use antibodies that specifically recognize phosphorylated forms at Ser18 and/or Ser80. These phospho-specific antibodies are essential for monitoring CEP4 activation status in response to PKC signaling .

• Species reactivity: Verify that the antibody recognizes CEP4 in your experimental species. Many validated antibodies target human CEP4, but cross-reactivity with mouse or rat homologs should be confirmed if working with these models .

• Application compatibility: Ensure the antibody is validated for your specific application (Western blotting, immunoprecipitation, immunofluorescence, etc.). For instance, when studying CEP4-binding partners, antibodies validated for immunoprecipitation under non-detergent conditions are crucial .

• Epitope location: Consider antibodies targeting epitopes outside the phosphorylation domains if you need to detect total CEP4 regardless of phosphorylation status.

• Monoclonal vs. polyclonal: Monoclonal antibodies offer high specificity for a single epitope, while polyclonal antibodies provide broader detection but potential cross-reactivity.

Research-based validation data should be reviewed before selecting an antibody for critical experiments.

How can researchers validate CEP4 antibody specificity in their experimental systems?

Validating CEP4 antibody specificity is crucial for generating reliable research data. Implement these methodological approaches for comprehensive validation:

• Positive and negative controls:

  • Positive control: Lysates from cells known to express CEP4 (e.g., MCF-10A cells)

  • Negative control: Lysates from cells with CEP4 knockdown using siRNA or shRNA

  • Recombinant CEP4 protein as a standard for antibody binding

• Phosphorylation-dependent detection verification:

  • Compare detection in lysates from PKC-activated cells (e.g., DAG-lactone treated) versus untreated cells

  • Use phosphorylation-resistant mutants (S18A, S80A, or S18A/S80A) alongside wild-type CEP4 to confirm phospho-specificity

• Western blot validation:

  • Verify a single band of expected molecular weight (~44 kDa for CEP4)

  • Perform peptide competition assays to confirm specificity

• Cross-technique validation:

  • Confirm consistent results across multiple techniques (Western blot, immunoprecipitation, immunofluorescence)

  • For phospho-specific antibodies, verify signal reduction following phosphatase treatment

• Mass spectrometry correlation:

  • When possible, validate antibody results against MS/MS phosphorylation site identification

Document all validation steps thoroughly for publication-quality research.

How can researchers effectively study the interaction between phosphorylated CEP4 and its binding partners?

To effectively study interactions between phosphorylated CEP4 and its binding partners, researchers should employ these sophisticated methodological approaches:

• Tandem Affinity Purification (TAP) method:

  • Generate TAP-tagged CEP4 constructs (wild-type and phosphomimetic mutants like S18D/S80D)

  • Perform sequential affinity chromatography under non-detergent conditions to preserve protein-protein interactions

  • Compare binding partners between phosphomimetic mutants (S18D/S80D) and phosphorylation-resistant mutants (S18A/S80A)

• Co-immunoprecipitation under native conditions:

  • Use non-detergent lysis buffers (e.g., 20 mM Tris, pH 7.4, 2 mM MgCl₂, 2 mM EGTA, 1 mM DTT) for cell disruption

  • Perform freeze-thaw cycles to gently release protein complexes

  • Immunoprecipitate using anti-FLAG antibodies for tagged CEP4 constructs

  • Validate interactions by Western blotting with antibodies against specific binding partners (e.g., TEM4, PARD6G)

• Proximity ligation assays:

  • Visualize CEP4-partner interactions in situ within cells

  • Compare signal patterns between wild-type and phosphomimetic mutants

  • Quantify interaction frequency in different cellular compartments

• Mass spectrometry analysis of binding partners:

  • Excise unique bands from SDS-PAGE of purified complexes

  • Perform MS/MS analysis for protein identification

  • Validate identified partners through orthogonal methods

Using these approaches, researchers have identified TEM4 (ARHGEF17) and PARD6G as binding partners of phosphorylated CEP4, providing insights into the mechanisms by which phospho-CEP4 promotes motility .

What techniques should be used to study the role of CEP4 phosphorylation in cell motility?

Studying CEP4 phosphorylation in cell motility requires a multifaceted approach combining molecular, cellular, and imaging techniques:

• Phosphomimetic and phosphorylation-resistant mutants:

  • Generate single-site mutants (S18D, S80D) and double mutants (S18D/S80D) to mimic phosphorylation

  • Create phosphorylation-resistant mutants (S18A, S80A, S18A/S80A) as controls

  • Express these constructs in weakly motile cells (e.g., MCF-10A) to assess functional outcomes

• Quantitative cell motility assays:

  • Transwell migration assays with defined pore sizes

  • Wound healing (scratch) assays with time-lapse imaging

  • Single-cell tracking to measure speed, persistence, and directionality

  • Compare motility between cells expressing different CEP4 variants

• Cytoskeletal visualization:

  • Stain cells with rhodamine-phalloidin to visualize F-actin structures

  • Quantify filopodia (microspikes) formation at leading edges

  • Analyze stress fiber patterns associated with different CEP4 phosphorylation states

• Small GTPase activation assays:

  • Perform pulldown assays using p21-activated kinase-binding domain-agarose beads to detect activated Rac

  • Compare Rac activation levels between cells expressing different CEP4 variants

  • Use small GTPase inhibitors (e.g., NSC23766 for Rac) to confirm pathway involvement

• Knockdown/rescue experiments:

  • Silence endogenous CEP4 using siRNA/shRNA

  • Rescue with siRNA-resistant wild-type or mutant CEP4 constructs

  • Assess functional recovery of motility phenotypes

Research has shown that phosphorylated CEP4 promotes motility through Rac activation, with phosphomimetic mutations leading to increased filopodia formation and enhanced cell movement .

How does CEP4 phosphorylation influence its interaction with Cdc42 and subsequent signaling?

CEP4 phosphorylation fundamentally alters its interaction with Cdc42, creating a molecular switch mechanism that redirects cellular signaling:

• Phosphorylation-dependent dissociation from Cdc42:

  • Unphosphorylated CEP4 binds to GTP-bound Cdc42 with high affinity

  • Phosphorylation at Ser18 and Ser80 dramatically reduces its affinity for constitutively active Cdc42 (Q61L)

  • Both in vitro binding assays and cellular co-immunoprecipitation experiments confirm this dissociation

• Quantitative binding characteristics:

  • When CEP4 is phosphorylated (or phosphomimetic S18D/S80D is expressed), co-immunoprecipitation studies show substantially lower amounts of bound Cdc42 compared to unphosphorylated (S18A/S80A) CEP4

  • This suggests a regulatory mechanism whereby PKC activity controls the CEP4-Cdc42 interaction

• Consequences for downstream signaling:

  CEP4 State	Cdc42 Binding	Downstream Effects	Cellular Outcome
  Unphosphorylated	High affinity	Canonical Cdc42 effector function	Normal, basal motility
  Phosphorylated (Ser18/Ser80)	Low affinity	1. Binding to TEM4 2. Rac activation 3. Actin reorganization	Enhanced cell motility with filopodia formation

• Alternative binding partners:

  • Upon dissociation from Cdc42, phosphorylated CEP4 forms complexes with TEM4 (ARHGEF17)

  • This phospho-CEP4/TEM4 complex promotes Rac activation, which is essential for the motility phenotype

  • Knockdown of TEM4 prevents both phospho-CEP4-induced Rac activation and the subsequent increase in cell motility

This phosphorylation-induced partner switching represents a novel regulatory mechanism for CEP4 function, where PKC activation redirects CEP4 from Cdc42-dependent pathways to alternative signaling mechanisms involving TEM4 and Rac .

What experimental protocols are optimal for detecting CEP4 phosphorylation in different cell types?

Detecting CEP4 phosphorylation requires careful consideration of cell type, stimulation conditions, and detection methods:

• Cell lysis and phosphoprotein preservation:

  • Lyse cells in buffer containing phosphatase inhibitors (e.g., 1% phosphatase inhibitor mixture)

  • Include PKC inhibitors (e.g., 10 μM bisindolylmaleimide) to prevent artificial phosphorylation during processing

  • Process samples quickly at 4°C to minimize dephosphorylation

• PKC stimulation protocols by cell type:

  • Epithelial cells (e.g., MCF-10A): Treat with DAG-lactone (cell-permeable PKC activator)

  • Fibroblasts: Phorbol ester (PMA) treatment (100 nM, 15-30 minutes)

  • Neuronal cells: Glutamate receptor activation (50 μM glutamate, 5 minutes)

  • Always include unstimulated controls to establish baseline phosphorylation

• Phosphorylation detection methods:

  • Western blotting: Use phospho-specific antibodies or PKC substrate antibodies

  • Phospho-specific enrichment: Immunoprecipitate with anti-FLAG (for tagged CEP4), then probe with PKC substrate antibody

  • Phos-tag SDS-PAGE: Enhanced separation of phosphorylated forms

  • Mass spectrometry: For precise site identification and quantification

• Protocol validation:

  • Use phosphorylation-resistant mutants (S18A/S80A) as negative controls

  • Include phosphomimetic mutants (S18D/S80D) as positive controls

  • Verify results with lambda phosphatase treatment to confirm phospho-specificity

For optimal results with MCF-10A cells, researchers have successfully used hypotonic, detergent-free lysis buffer (20 mM Tris, pH 7.4, 2 mM MgCl₂, 2 mM EGTA, 1 mM DTT, 10 μM bisindolylmaleimide, 0.1% protease inhibitor mixture, 1% phosphatase inhibitor mixture) with freeze-thaw cycles to preserve protein interactions .

What are the recommended controls for CEP4 antibody experiments?

Implementing appropriate controls is essential for generating reliable data with CEP4 antibodies:

• Positive controls:

  • Cell lysates known to express CEP4 (e.g., MCF-10A cells)

  • Recombinant CEP4 protein

  • Cells transfected with FLAG-tagged or TAP-tagged CEP4

• Negative controls:

  • CEP4 knockdown cells (siRNA or shRNA treatment)

  • Cell lines not expressing CEP4

  • Vector control (VC) transfections for comparison with CEP4 mutants

  • Primary antibody omission control

• Phosphorylation-specific controls:

  • Phosphorylation-resistant mutants (S18A, S80A, S18A/S80A)

  • Phosphomimetic mutants (S18D, S80D, S18D/S80D)

  • PKC activator (DAG-lactone) treatment versus untreated cells

  • Lambda phosphatase treatment to remove phosphorylation

• Specificity controls:

  • Peptide competition assays

  • Secondary antibody-only controls

  • Isotype control antibodies

• Functional controls:

  • GTPase inhibitors (e.g., NSC23766 for Rac)

  • Partner protein knockdown (e.g., TEM4 shRNA)

  • Scrambled control (SC) shRNA for comparison with specific knockdowns

Research has demonstrated the importance of these controls in validating the specificity of observed effects. For example, the phosphorylation-resistant double mutant (S18A/S80A) effectively blocked the phosphorylation signal detected by the PKC substrate antibody in DAG-lactone-treated cells, confirming the specificity of the phosphorylation sites .

How should researchers troubleshoot inconsistent results with CEP4 antibodies?

When facing inconsistent results with CEP4 antibodies, implement this systematic troubleshooting approach:

• Antibody-specific issues:

  • Validate antibody lot: Different lots may have varying specificity profiles

  • Optimize antibody concentration: Test a range of dilutions (1:500 to 1:5000)

  • Adjust incubation conditions: Try different temperatures (4°C overnight vs. room temperature) and times

  • Test multiple antibodies: Use antibodies targeting different epitopes of CEP4

• Sample preparation challenges:

  • Phosphorylation preservation: Ensure phosphatase inhibitors are fresh and effective

  • Protein degradation: Add protease inhibitors; minimize freeze-thaw cycles

  • Protein solubility: For membrane-associated complexes, test different lysis buffers

  • Incomplete denaturation: Adjust SDS concentration and heating conditions

• Technical considerations for specific applications:

  Application	Common Issues	Troubleshooting Approach
  Western blot	Background bands	1. Increase blocking time/concentration 2. Wash more extensively 3. Use different blocking agent
  Immunoprecipitation	Poor pull-down efficiency	1. Use non-detergent conditions for complex preservation 2. Adjust bead amount and incubation time 3. Try different antibody orientation (direct vs. indirect IP)
  Immunofluorescence	Weak signal	1. Optimize fixation method 2. Test antigen retrieval 3. Increase antibody concentration

• Biological variability:

  • Cell density effects: Standardize cell confluence at collection

  • Cell cycle influence: Synchronize cells if necessary

  • PKC activation state: Control stimulation conditions carefully

  • Binding partner interference: Consider detergent conditions that may disrupt complexes

• Controls for interpretation:

  • Use phosphomimetic (S18D/S80D) and phosphorylation-resistant (S18A/S80A) mutants as standards to interpret ambiguous signals

  • Include positive cellular controls where phosphorylation is known to occur (e.g., DAG-lactone-treated cells)

When troubleshooting experiments involving CEP4-partner interactions, remember that detergent-free conditions are critical for preserving these complexes during immunoprecipitation or TAP procedures .

How can researchers effectively use CEP4 antibodies to study the role of TEM4 in cell motility?

Studying the CEP4-TEM4 relationship in cell motility requires sophisticated experimental approaches using CEP4 antibodies:

• Co-localization studies:

  • Perform double immunofluorescence using CEP4 and TEM4 antibodies

  • Analyze co-localization at the leading edge of motile cells

  • Compare wild-type CEP4 versus phosphomimetic (S18D/S80D) mutants

  • Quantify Pearson's correlation coefficient under different conditions

• Co-immunoprecipitation optimization:

  • Use non-detergent lysis conditions to preserve the CEP4-TEM4 complex

  • Implement tandem affinity purification with TAP-tagged CEP4 constructs

  • Compare binding partner profiles between phosphorylation states

  • Validate interactions through reverse co-immunoprecipitation with TEM4 antibodies

• Functional interaction analysis:

  • Silence TEM4 using GFP-shRNA constructs while expressing CEP4 phosphomutants

  • Measure cell motility and Rac activation to determine functional dependency

  • Compare motility of cells expressing phosphomimetic CEP4 with and without TEM4 knockdown

  • Quantify filopodia formation as a downstream measure of pathway activation

• Mechanistic dissection using domain mutants:

  • Generate domain-specific mutations in either CEP4 or TEM4

  • Identify critical domains required for the CEP4-TEM4 interaction

  • Assess whether GEF activity of TEM4 is required for phospho-CEP4-mediated motility

• Pathway validation with inhibitors:

  • Use Rac inhibitor (NSC23766) to block downstream signaling

  • Combine with phospho-CEP4 expression to determine pathway dependency

  • Quantify both motility and cytoskeletal changes

Research has demonstrated that TEM4 knockdown significantly reduces (by almost 40%) the motility induced by phosphomimetic D/D-CEP4 mutant, confirming TEM4's essential role in phospho-CEP4-mediated motility .

What approaches can researchers use to analyze the impact of CEP4 phosphorylation on Rac activation and actin dynamics?

To analyze the relationship between CEP4 phosphorylation, Rac activation, and actin dynamics, researchers should implement these methodological approaches:

• Rac activation assays:

  • Perform pulldown assays using p21-activated kinase-binding domain-agarose beads

  • Compare Rac-GTP levels in cells expressing different CEP4 variants

  • Quantify activation using Western blotting with Rac-specific antibodies

  • Establish temporal dynamics by creating activation time courses

• Live-cell imaging of actin dynamics:

  • Transfect cells with fluorescent actin markers (LifeAct-GFP)

  • Perform time-lapse microscopy to track filopodia formation and dynamics

  • Quantify parameters including protrusion rate, persistence, and number

  • Compare cells expressing phosphomimetic versus phosphorylation-resistant CEP4

• Cytoskeletal staining and quantification:

  • Fix cells and stain with rhodamine-phalloidin to visualize F-actin

  • Conduct quantitative analysis of:

    • Filopodia/microspike numbers at leading edges

    • Stress fiber patterns and abundance

    • Actin network architecture differences

  • Compare statistical differences between CEP4 variant populations

• Inhibitor studies:

  • Apply Rac-specific inhibitor (NSC23766) to cells expressing phosphomimetic CEP4

  • Document the elimination of actin-based protrusions

  • Correlate structural changes with functional motility inhibition

  • Establish dose-response relationships

• Upstream-downstream validation:

  • Silence TEM4 using shRNA and measure effects on:

    • Rac activation levels

    • Actin-based protrusions

    • Cell motility

  • This approach confirms the sequential CEP4→TEM4→Rac→actin pathway

Research has demonstrated that cells expressing phosphomimetic D/D-CEP4 develop prominent actin-based filopodia at the leading edge in >80% of transfectants, compared to just 10% in cells expressing the phosphorylation-resistant A/A-CEP4 mutant. These cellular protrusions were completely eliminated by Rac inhibitor treatment, confirming the Rac dependency of this phenotype .

What are the known limitations of current CEP4 antibodies and how might they be addressed?

Current CEP4 antibodies face several limitations that researchers should consider, along with potential solutions:

• Phospho-state specificity challenges:

  • Limitation: Many available antibodies cannot distinguish between phosphorylated and unphosphorylated CEP4

  • Solution: Develop highly specific phospho-antibodies targeting Ser18 and Ser80 individually and in combination

  • Approach: Use phosphopeptide immunization strategies with careful negative selection

• Cross-reactivity with other CEP family members:

  • Limitation: Potential cross-reactivity with other CEP isoforms due to sequence homology

  • Solution: Target unique regions of CEP4 that differ from other family members

  • Validation: Confirm specificity using knockout/knockdown models and recombinant protein panels

• Limited availability of application-specific antibodies:

  • Limitation: Many antibodies work for Western blot but not for immunoprecipitation or immunofluorescence

  • Solution: Generate new antibodies optimized for specific applications

  • Strategy: Use native protein immunization for antibodies intended for immunoprecipitation

• Detection challenges in complex samples:

  • Limitation: Difficulty detecting endogenous CEP4 in tissues with low expression

  • Solution: Develop signal amplification protocols compatible with CEP4 antibodies

  • Approach: Implement proximity ligation assays for enhanced sensitivity

• Technical issues for studying CEP4-protein complexes:

  • Limitation: Standard detergent conditions disrupt CEP4-partner interactions

  • Solution: Optimize native, non-detergent lysis conditions that preserve complexes

  • Evidence: Non-detergent conditions have been successful in identifying TEM4 as a CEP4 binding partner

These limitations may be addressed through the development of next-generation antibodies with enhanced specificity, sensitivity, and application versatility for CEP4 research.

What are promising future research directions for CEP4 antibody applications?

Future CEP4 antibody applications hold significant promise for advancing our understanding of cell signaling and motility mechanisms:

• Spatiotemporal dynamics of CEP4 phosphorylation:

  • Develop FRET-based biosensors using CEP4 antibody fragments

  • Create live-cell imaging tools to visualize CEP4 phosphorylation in real-time

  • Map the subcellular locations of CEP4 phosphorylation during migration

• CEP4 in disease models:

  • Investigate CEP4 phosphorylation status in cancer progression models

  • Examine correlation between CEP4 phosphorylation and metastatic potential

  • Develop tissue microarray applications using phospho-specific antibodies

• Therapeutic targeting of CEP4 pathways:

  • Identify small molecules that modulate the CEP4-TEM4 interaction

  • Screen for compounds that alter CEP4 phosphorylation states

  • Develop antibody-based targeted approaches to disrupt specific CEP4 complexes

• Systems-level analysis of CEP4 interaction networks:

  • Apply proximity-dependent biotinylation (BioID) with CEP4 antibodies

  • Map the complete interactome of phosphorylated versus unphosphorylated CEP4

  • Identify context-dependent interaction partners in different cell types

• CEP4 in cytoskeletal remodeling mechanisms:

  • Develop super-resolution microscopy applications using CEP4 antibodies

  • Investigate CEP4's role in specialized cellular structures

  • Examine the detailed molecular architecture of CEP4-containing complexes at leading edges

These future directions leverage the specificity of CEP4 antibodies to explore fundamental questions about cell motility regulation and potential therapeutic applications targeting cell migration in disease states.

What are the key considerations for researchers working with CEP4 antibodies?

Researchers working with CEP4 antibodies should prioritize several critical considerations to ensure experimental success and meaningful results:

• Phosphorylation state awareness: Recognize that CEP4 function dramatically changes depending on its phosphorylation status at Ser18 and Ser80. Select antibodies appropriate for detecting the specific CEP4 state relevant to your research question .

• Preservation of protein interactions: When studying CEP4 binding partners, employ non-detergent lysis conditions to maintain native protein complexes. Standard detergent-based protocols may disrupt crucial interactions, particularly with partners like TEM4 .

• Functional validation approach: Combine antibody-based detection with functional studies using phosphomimetic (S18D/S80D) and phosphorylation-resistant (S18A/S80A) mutants to establish mechanistic connections between phosphorylation and cellular outcomes .

• Context dependence: Consider that CEP4 functions may vary by cell type, stimulation conditions, and microenvironment. What is observed in one cellular system may not directly translate to another.

• Pathway integration: Recognize that CEP4 operates within a complex signaling network involving PKC, Cdc42, TEM4, and Rac. Comprehensive analysis requires examining these pathway components together rather than in isolation .

By addressing these considerations, researchers can more effectively leverage CEP4 antibodies to uncover the complex regulatory mechanisms controlling cell motility and cytoskeletal dynamics.

How does our understanding of CEP4 phosphorylation contribute to broader cell motility research?

The discovery of CEP4's phosphorylation-dependent functions provides significant insights into cell motility regulation with broader implications:

• Molecular switch mechanism: CEP4 phosphorylation represents a prototypical molecular switch where phosphorylation redirects protein function through altered binding partner preferences. This dissociation from Cdc42 and association with TEM4 demonstrates how post-translational modifications can completely rewire signaling pathways .

• Integration of PKC and Rho GTPase signaling: CEP4 phosphorylation establishes a direct mechanistic link between PKC activation and Rho GTPase signaling, connecting two major regulatory systems controlling cell migration .

• Novel pathway for filopodia formation: The phospho-CEP4→TEM4→Rac pathway reveals an alternative mechanism for filopodia formation that operates independently of the canonical Cdc42 pathway, expanding our understanding of cytoskeletal regulation .

• Potential therapeutic implications: Understanding the molecular details of phospho-CEP4-mediated motility provides potential targets for modulating cell migration in pathological contexts like cancer metastasis.

• Methodological advances: The approaches developed to study CEP4 phosphorylation (TAP methods, phosphomutant analysis, non-detergent complex preservation) provide valuable tools applicable to studying other phosphorylation-regulated proteins .