Phospho-KIF2C (S95) Antibody

What is Phospho-KIF2C (S95) Antibody and what cellular functions does it help investigate?

Phospho-KIF2C (S95) Antibody is a polyclonal antibody that specifically detects endogenous levels of human KIF2C protein only when phosphorylated at serine 95 . KIF2C (also known as MCAK - Mitotic Centromere-Associated Kinesin) is a microtubule-depolymerizing kinesin that plays critical roles in multiple cellular processes including:

• Microtubule dynamics regulation and depolymerization

• Cell cycle progression, particularly during mitosis

• Chromosome segregation during cell division

• DNA double-strand break (DSB) repair and genomic stability

• Synaptic plasticity and neuronal function

The antibody enables researchers to specifically investigate the phosphorylation state of KIF2C at S95, which represents an important regulatory modification that affects KIF2C's functions in different cellular contexts .

What are the optimal experimental conditions for Western Blot analysis using Phospho-KIF2C (S95) Antibody?

For optimal Western Blot results using Phospho-KIF2C (S95) Antibody:

Sample Preparation:

• Use fresh tissue or cell lysates treated with phosphatase inhibitors to preserve phosphorylation status

• TNF treatment (10ng/ml for 30 minutes) has been demonstrated to enhance phosphorylation signal in HeLa cells

• Expected molecular weight of KIF2C is approximately 80-81 kDa

Protocol Optimization:

• Use dilution range of 1:500-1:2000 in blocking buffer containing 5% BSA (not milk)

• Include appropriate positive controls (cells with known KIF2C phosphorylation status)

• Include negative controls by:

  • Pre-incubating antibody with immunizing peptide

  • Using phosphatase-treated samples

• For validation, observe band disappearance in blocking peptide competition assay

Critical Considerations:

• Store antibody at -20°C and avoid repeated freeze-thaw cycles

• Block with BSA rather than milk to prevent interference with phospho-epitope recognition

• Include appropriate loading controls for normalization (total KIF2C or housekeeping proteins)

How can researchers effectively use Phospho-KIF2C (S95) Antibody to study DNA damage response mechanisms?

Studies have established KIF2C as a critical player in DNA double-strand break (DSB) repair . To effectively study KIF2C's role in DNA damage response:

Experimental Design:

• Recruitment Kinetics Analysis:

  • Induce DNA damage using etoposide, ionizing radiation, or laser microirradiation

  • Perform time-course immunofluorescence (IF) staining (1:200-1:1000 dilution)

  • Co-stain with established DSB markers (γ-H2AX, 53BP1)

  • Note: KIF2C recruitment to DNA damage sites is dependent on both PARP and ATM activities

• Functional Assays:

  • Utilize KIF2C knockdown/knockout in combination with Phospho-KIF2C (S95) antibody to:

    • Measure DSB repair efficiency using γ-H2AX kinetics or comet assay

    • Track DSB mobility using live-cell imaging with GFP-53BP1 foci

    • Assess DSB repair pathway choice (NHEJ vs. HR)

• Mechanistic Studies:

  • Investigate whether S95 phosphorylation affects:

    • DNA damage foci formation and resolution

    • DSB mobility (KIF2C depletion reduces DSB mobility)

    • Interaction with repair proteins like PARP1, H2AX, and Ku70/80

Important Finding: KIF2C depletion, or inhibition of its microtubule depolymerase activity, reduces DSB mobility, impairs DNA damage foci formation, and decreases foci fusion and resolution , suggesting a critical role for phosphorylated KIF2C in facilitating DNA damage repair.

How does phosphorylation at S95 regulate KIF2C's function in oncogenic pathways and cancer progression?

KIF2C has been identified as a potential oncogene in multiple cancer types. Studies utilizing phospho-specific antibodies have revealed important insights into how S95 phosphorylation may influence oncogenic functions:

Hepatocellular Carcinoma (HCC):

Pancreatic Ductal Adenocarcinoma (PDAC):

• KIF2C facilitates tumor growth and metastasis

• Cell cycle analysis shows abnormal proliferation in G2 and S phases in KIF2C-overexpressing cells

Investigation Approaches:

• Use Phospho-KIF2C (S95) antibody to compare phosphorylation status between:

  • Tumor vs. adjacent normal tissues

  • Early vs. advanced stage tumors

  • Primary vs. metastatic lesions

• Correlate S95 phosphorylation levels with:

  • Activation of downstream signaling pathways (MEK/ERK, PI3K/Akt)

  • Cell cycle progression markers

  • EMT markers (E-cadherin, Vimentin, Snail)

• Investigate whether S95 phosphorylation affects KIF2C's:

  • Subcellular localization

  • Protein stability and turnover

  • Interaction with oncogenic signaling molecules

This research has significant translational potential, as KIF2C is anticipated to serve as a biomarker for cancer diagnosis, prognosis, and potentially as a target for therapy .

What is known about the kinases responsible for S95 phosphorylation of KIF2C and how can these be studied?

The regulation of KIF2C through phosphorylation is complex and involves several kinases. While the search results don't specifically identify which kinases phosphorylate S95, information from related phosphorylation sites provides insights:

Potential Kinases and Regulatory Mechanisms:

• Aurora B kinase (AURKB) regulates KIF2C association with centromeres/kinetochores and its microtubule depolymerization activity through phosphorylation

• DNA damage response kinases like ATM may be involved, as KIF2C is "phosphorylated upon DNA damage"

• PKA has been identified in phosphorylation networks that may include KIF2C

Methodological Approaches to Study Kinase-KIF2C Relationships:

• In vitro Kinase Assays:

  • Incubate recombinant KIF2C with candidate kinases (AURKB, ATM, PKA)

  • Use Phospho-KIF2C (S95) antibody to detect specific phosphorylation

  • Compare with phospho-deficient mutants (S95A)

• Kinase Inhibitor Studies:

  • Treat cells with specific kinase inhibitors (Aurora kinase inhibitors, ATM inhibitors)

  • Monitor S95 phosphorylation status using the antibody

  • Note: KIF2C recruitment to DNA damage sites is dependent on both PARP and ATM activities

• Mass Spectrometry-Based Approaches:

  • Use the M3 (Motif discovery based on Microarray and MS/MS) approach described in search result

  • Identify position weight matrices (PWMs) that might predict kinases for S95

  • Validate predictions using targeted experiments with Phospho-KIF2C (S95) antibody

• Functional Analysis:

  • Create phospho-mimetic (S95D/E) and phospho-deficient (S95A) KIF2C mutants

  • Compare their microtubule depolymerization activity

  • Assess cellular localization and protein-protein interactions

Understanding the kinases responsible for S95 phosphorylation could provide valuable insights into how KIF2C function is regulated in different physiological and pathological contexts.

How can researchers validate the specificity of Phospho-KIF2C (S95) Antibody in their experimental systems?

Validation of phospho-specific antibodies is crucial for reliable results. For Phospho-KIF2C (S95) Antibody, consider these validation approaches:

Recommended Validation Methods:

• Peptide Competition Assay:

  • Pre-incubate antibody with the immunizing phosphopeptide

  • Signal should be significantly reduced or eliminated in Western blot or IF

  • Example: Results show signal elimination when pre-incubated with immunizing peptide in A549 cells

• Phosphatase Treatment:

  • Treat half of your sample with lambda phosphatase

  • Compare with untreated sample - signal should be absent in phosphatase-treated sample

• Genetic Manipulation:

  • Use KIF2C knockout or knockdown cells as negative controls

  • Reintroduce wild-type KIF2C or phospho-mutant (S95A) for rescue experiments

  • Phospho-signal should disappear with S95A mutant

• Induction of Phosphorylation:

  • Treat cells with known inducers of DNA damage (as KIF2C is phosphorylated upon DNA damage)

  • Example: TNF treatment (10ng/ml for 30 minutes) enhances phosphorylation signal in HeLa cells

• Cross-Validation with Different Antibodies or Methods:

  • Compare results with another phospho-specific antibody if available

  • Use mass spectrometry to confirm phosphorylation status

Proper validation ensures that the observed signals truly represent phosphorylated KIF2C at S95 rather than non-specific binding or artifacts.

What are common challenges in experiments using Phospho-KIF2C (S95) Antibody and how can they be addressed?

Researchers often encounter several challenges when working with phospho-specific antibodies like Phospho-KIF2C (S95) Antibody:

Challenge 1: Low Signal-to-Noise Ratio

• Solution:

  • Optimize antibody dilution (start with 1:500 for WB, 1:200 for IF)

  • Use fresh samples with phosphatase inhibitors

  • Increase protein loading for WB (50-100 μg)

  • Use signal enhancement systems (e.g., TSA for IF)

Challenge 2: Phosphorylation Status Changes During Sample Processing

• Solution:

  • Immediately add phosphatase inhibitors during sample collection

  • Keep samples on ice and process quickly

  • Use phospho-preserving lysis buffers

  • Consider fixation methods that better preserve phospho-epitopes

Challenge 3: Variability in KIF2C Expression Levels Between Samples

• Solution:

  • Always normalize phospho-KIF2C to total KIF2C levels

  • Use quantitative analysis software

  • Include appropriate loading controls

  • Run biological replicates (minimum n=3)

Challenge 4: Cell Cycle-Dependent Phosphorylation Variability

• Solution:

  • Synchronize cells (KIF2C associates with centromeres at early prophase and remains there until after telophase)

  • Perform cell cycle analysis in parallel

  • Use cell cycle markers to correlate with KIF2C phosphorylation

Challenge 5: Cross-Reactivity with Other Phosphorylated Proteins

• Solution:

  • Always confirm band size (expected MW: 80-81 kDa)

  • Include KIF2C knockout/knockdown controls

  • Perform peptide competition assays

These methodological approaches will help ensure reliable and reproducible results when using Phospho-KIF2C (S95) Antibody in various research applications.

How can Phospho-KIF2C (S95) Antibody be used to investigate KIF2C's role in neurodegenerative disorders?

Recent research has revealed that KIF2C plays a critical role in neuronal function through regulating microtubule dynamics in dendrites and synapses . This opens new research avenues using Phospho-KIF2C (S95) Antibody:

Neuronal Research Applications:

• Synaptic Plasticity Studies:

  • KIF2C regulates spine morphology and synaptic transmission

  • Use Phospho-KIF2C (S95) Antibody to:

    • Determine if S95 phosphorylation changes during synaptic activity

    • Correlate phosphorylation with changes in dendritic spine morphology

    • Assess co-localization with synaptic markers using IF (1:200-1:1000 dilution)

• Neurodegeneration Models:

  • Many neurodegenerative disorders feature microtubule dysfunction

  • Investigate whether:

    • S95 phosphorylation is altered in Alzheimer's, Parkinson's, or ALS models

    • KIF2C phosphorylation correlates with cognitive decline in animal models

    • Manipulation of KIF2C phosphorylation affects neurodegeneration progression

• Mechanistic Studies:

  • Determine if S95 phosphorylation influences:

    • Microtubule invasion of spines (which KIF2C regulates in an activity-dependent manner)

    • AMPA receptor membrane expression (which KIF2C deficiency increases)

    • Long-term potentiation (which KIF2C deficiency impairs)

Methodological Approaches:

• Use primary neuron cultures and brain tissue samples for WB and IF

• Combine Phospho-KIF2C (S95) antibody staining with neuronal markers

• Perform live imaging studies of spine dynamics in conjunction with fixed-cell phospho-KIF2C analysis

• Utilize conditional KIF2C knockout mice for in vivo studies

This research direction could provide significant insights into how defects in microtubule dynamics contribute to synaptic dysfunction and neurodegeneration.

What is the potential of KIF2C as a therapeutic target and how can Phospho-KIF2C (S95) Antibody facilitate drug development?

Given KIF2C's roles in cancer progression and its recently discovered functions in neuronal plasticity , it represents a promising therapeutic target. Phospho-KIF2C (S95) Antibody can facilitate drug development in several ways:

Cancer Therapeutics Development:

• Target Validation:

  • Use the antibody to assess S95 phosphorylation status in:

    • Patient-derived tumor samples

    • Cancer cell lines before and after treatment

    • In vivo xenograft models

  • Correlation with clinical outcomes provides validation for targeting this pathway

• High-Throughput Screening:

  • Develop cell-based assays using the antibody to screen for compounds that:

    • Inhibit S95 phosphorylation

    • Alter KIF2C localization or function

    • Specifically target cancer cells with high phospho-KIF2C levels

• Mechanism-Based Drug Design:

  • Target the ATP-binding domain that affects KIF2C's microtubule depolymerization activity

  • Several studies have found that KIF2C's depolymerase activity is critical for:

    • DNA repair

    • Cancer cell proliferation

    • Neuronal function

  • The G491A mutant (human G495A) lacking ATP hydrolysis/MT depolymerization activity serves as a model

Biomarker Applications:

• Use Phospho-KIF2C (S95) Antibody for:

  • Patient stratification in clinical trials

  • Monitoring treatment response

  • Early detection of recurrence

  • Correlation with resistance mechanisms

Key Considerations for Drug Development:

• Target tissue specificity (e.g., cancer vs. neurons)

• Differential expression in tissues (high in thymus and testis, low in intestine and colon)

• Potential off-target effects on normal cell division

• Combined inhibition strategies with other cancer pathways

Recent in silico studies have identified potential small molecules that may bind to KIF2C's active site , providing a starting point for drug development efforts that could be validated using the Phospho-KIF2C (S95) Antibody.