CDC14A Antibody

What is CDC14A and why is it important in cellular research?

CDC14A (Cell Division Cycle 14 homolog A) is a dual-specificity phosphatase that dephosphorylates proteins at tyrosine or serine/threonine residues. It plays critical roles in cell cycle regulation by activating mitotic exit through dephosphorylation of cyclin-dependent kinase substrates . While initially thought to be essential for cell cycle progression like its yeast counterpart, human CDC14A has revealed additional functions in cell migration, adhesion, actin cytoskeleton organization, and cilia regulation . Its clinical significance stems from its downregulation in various cancers, including colorectal carcinoma, which correlates with poor prognosis .

What types of CDC14A antibodies are available for research applications?

Several types of CDC14A antibodies are available for research:

Different antibodies recognize specific regions of CDC14A and vary in their applications, making selection critical for experimental success .

What is the subcellular localization pattern of CDC14A, and how can this impact antibody selection?

CDC14A exhibits dynamic subcellular localization patterns that influence antibody selection:

• Sequestered in the nucleolus until release during mitosis

• Associates with F-actin filaments at the cell leading edge

• Localizes to the basal body in ciliated cells, with the most intense signal at the proximal end containing the linker protein C-Nap1

• Shows two distinct pools at the basal body: one at the proximal end and a weaker signal near the distal end

When selecting antibodies, researchers should consider whether their target localization requires antibodies that recognize specific conformational states, as some antibodies (like those in source ) recognize conformation-specific antigens and fail to detect the protein in immunoblots while working in immunofluorescence studies .

What are the optimal experimental conditions for using CDC14A antibodies in Western blot applications?

For optimal Western blot results with CDC14A antibodies:

• Sample preparation: Extract proteins using RIPA or NP-40 buffer with protease inhibitors and phosphatase inhibitors (especially important since CDC14A is a phosphatase)

• Loading amount: 20-40 μg of total protein per lane is recommended

• Dilution ratios:

  • For polyclonal antibodies (e.g., 13660-1-AP): 1:500-1:2000

  • For monoclonal antibodies (e.g., MAB4457): 1 μg/mL

• Expected molecular weight: CDC14A appears at approximately 66-70 kDa

• Reducing conditions: Use reducing conditions with immunoblot buffer group 1

• Detection systems: Both HRP-conjugated secondary antibodies and fluorescence-based systems are compatible

When troubleshooting, note that some cell lines show stronger expression, with HepG2, Raji, and C2C12 cells demonstrating reliable detection .

How can I optimize immunofluorescence protocols to detect endogenous CDC14A?

For successful immunofluorescence detection of endogenous CDC14A:

• Fixation method: Use 100% methanol at -20°C for 20 minutes (critical for preserving CDC14A epitopes)

• Antibody selection: Use antibodies validated for IF applications; some recognize conformation-specific epitopes better than others

• Dilution ranges:

  • Polyclonal antibodies: 0.25-2 μg/mL

  • Monoclonal antibodies: Verify specific recommendations

• Co-staining markers:

  • For centrosome studies: γ-tubulin

  • For actin colocalization: Phalloidin conjugates

  • For basal body: CEP164 (distal appendage) or ODF2/ninein (subdistal appendages)

• Signal amplification: Consider tyramide signal amplification for weak signals

• Negative controls: Use CDC14A knockout cells to confirm specificity

The dynamic localization of CDC14A requires careful timing in synchronized cells to capture specific patterns .

What methodologies are effective for studying CDC14A phosphatase activity in vitro and in vivo?

To study CDC14A phosphatase activity:

In vitro approaches:

• Recombinant protein assays: Use purified CDC14A fusion proteins (like Ag4969 ) with artificial substrates (p-nitrophenyl phosphate) or known substrates

• Phosphatase activity assays: Monitor dephosphorylation of 32P-labeled substrates

• Phospho-peptide arrays: Identify substrate preferences

In vivo approaches:

• CDC14A phosphatase-dead (PD) cell lines: Generate C278S mutant lines that lack phosphatase activity but retain binding capacity

• Mass spectrometry-based phosphoproteomics: Compare phosphorylation profiles between CDC14A-overexpressing and control cells

• Proximity-dependent biotin identification (BioID): Fuse CDC14A to BirA to identify proximal substrates

• siRNA knockdown: Validate with RT-PCR due to antibody limitations

Studies have identified several CDC14A substrates, including actin regulators like eplin, which is dephosphorylated at serine residues 362, 374, 604, and 609 .

Why might CDC14A antibodies show inconsistent results between Western blot and immunofluorescence applications?

Inconsistencies between Western blot and immunofluorescence results for CDC14A antibodies occur for several reasons:

• Conformation-dependent epitopes: Some CDC14A antibodies (as noted in source ) recognize conformation-specific antigens that are preserved in immunofluorescence but destroyed during protein denaturation for Western blotting

• Cross-reactivity profiles: Antibodies may detect different epitopes with varying specificity:

  • Anti-hCDC14A antibodies have been reported to detect weak nuclear signals that may arise from cross-reactivity with nuclear proteins in CDC14A knockout cells

• Post-translational modifications: CDC14A phosphorylation state can affect antibody recognition

• Isoform specificity: Human CDC14A has multiple isoforms that antibodies may detect differentially

For troubleshooting inconsistencies:

• Validate antibodies using CDC14A knockout cells as negative controls

• Use multiple antibodies targeting different epitopes

• Consider alternative detection methods like RT-PCR when antibody detection is challenging

How can researchers distinguish between CDC14A and its paralog CDC14B in experimental systems?

Distinguishing between CDC14A and CDC14B requires careful experimental design:

• Antibody selection:

  • Use antibodies raised against the divergent C-terminus of CDC14A, as this region differs substantially from CDC14B

  • Validate antibody specificity using recombinant proteins of both paralogs

• RNA analysis:

  • Design isoform-specific primers for RT-PCR analysis

  • Example: CDC14A siRNAs (siRNA 1) have been validated for specificity against CDC14A without affecting CDC14B transcripts

• Localization patterns:

  • CDC14A: Associates with actin filaments, basal body, and centrosomes

  • CDC14B: Predominantly nucleolar with distinct localization pattern

• Functional analysis:

  • CDC14A: Primarily regulates actin dynamics, cell migration, and adhesion

  • CDC14B: Different functional profile

• Knockout validation:

  • Use CRISPR/Cas9-generated knockout cells specific to each paralog

When publishing results, clearly specify which paralog was studied and provide validation data demonstrating specificity of detection methods.

How should researchers interpret CDC14A localization patterns in relation to its biological functions?

CDC14A localization provides important insights into its functions:

• Actin filament association:

  • Indicates role in regulating actin dynamics

  • Correlates with functions in cell migration and adhesion

  • CDC14A overexpression induces stress fiber formation, while CDC14A-PD cells show decreased stress fibers

• Leading edge localization:

  • Co-localization with KIBRA at leading edge suggests functional relationship

  • Consistent with role in cell migration regulation

  • Impaired CDC14A activity increases cell migration and reduces cell-cell adhesion

• Basal body localization:

  • Two distinct pools at basal bodies indicate function in cilia regulation

  • Proximal pool (intense signal): Contains C-Nap1 linker protein

  • Distal pool (weaker signal): Underneath CEP164, overlapping with ODF2/ninein

  • CDC14A modulates primary cilium length

• Cell cycle-dependent changes:

  • Sequestered in nucleolus until mitosis

  • Relates to historical role in cell cycle regulation

When analyzing CDC14A localization, consider how patterns change in response to stimuli (e.g., EGF treatment, which induces actin cytoskeleton rearrangements that CDC14A counteracts ).

What are the implications of CDC14A dysregulation in disease contexts, and how can antibodies help understand these mechanisms?

CDC14A dysregulation has significant disease implications that can be investigated using antibodies:

• Cancer progression:

  • Downregulation of CDC14A mRNA correlates with colorectal carcinoma and poor prognosis

  • CDC14A PD cells show enhanced migration and reduced adhesion, resembling metastatic phenotypes

  • Antibodies can be used for tissue microarray analysis to correlate CDC14A expression with clinical outcomes

• Genomic instability:

  • CDC14A dysfunction leads to impaired centrosome separation and chromosome segregation

  • Immunofluorescence with CDC14A antibodies can assess centrosome abnormalities

• Hearing loss and infertility:

  • CDC14A is essential for hearing and male fertility in mammals

  • CDC14A antibodies can help characterize expression in specialized tissues

• Cell adhesion defects:

  • CDC14A PD leads to downregulation of E-cadherin and reduction in α/β-catenin at cell-cell adhesions

  • Double immunofluorescence with CDC14A and adhesion markers provides mechanistic insights

Therapeutic relevance: Understanding CDC14A's regulation of tumor-relevant pathways (like KIBRA/Hippo) suggests potential for targeted interventions in cancers with dysregulated CDC14A .

How can CDC14A antibodies be utilized in phosphoproteomics workflows to identify novel substrates?

Integrating CDC14A antibodies into phosphoproteomics workflows enables discovery of novel substrates:

• Substrate-trapping approaches:

  • Use antibodies against catalytically inactive CDC14A-C278S mutant, which functions as a substrate trap

  • Immunoprecipitate CDC14A-C278S complexes and identify interacting proteins by mass spectrometry

  • This approach has successfully identified eplin as a CDC14A substrate

• Comparative phosphoproteomics:

  • Compare phosphopeptide profiles between CDC14A-overexpressing and control cells

  • Example methodology from source :

    • Culture cells in SILAC media (heavy/light)

    • Induce CDC14A expression in heavy-labeled cells

    • Mix extracts 1:1, perform phosphopeptide enrichment

    • Analyze by LC-MS/MS

  • This approach identified 68 hypophosphorylated sites (0.5% of detected phosphosites) in CDC14A-expressing cells

• Proximity-dependent approaches:

  • Fuse BirA to CDC14A to biotinylate proximal proteins

  • Identify biotinylated proteins using streptavidin pulldown and mass spectrometry

  • Source used this approach to identify actin-associated proteins as CDC14A substrates

• Consensus motif analysis:

  • CDC14A dephosphorylates predominantly pSP motifs (95.6% P-serine, 4.4% P-threonine)

  • In cycling cells, ~40% of sites follow pSPxK/R pattern; under serum starvation, preference for positively charged amino acids at pS+3 drops to 28%

These approaches have identified actin regulators including drebrin (DBN1), synaptopodin (SYNPO), and LIMA1 as CDC14A substrates .

What emerging techniques are advancing our understanding of CDC14A's role in actin cytoskeleton organization?

Cutting-edge techniques are revealing CDC14A's role in actin dynamics:

• Live-cell imaging with engineered CDC14A biosensors:

  • Fluorescent protein-tagged CDC14A variants

  • FRET-based activity sensors to monitor real-time phosphatase activity

  • These approaches reveal dynamic association with actin during cell migration

• Genome engineering approaches:

  • CRISPR/Cas9-generated CDC14A phosphatase-dead (PD) cell lines

  • Phenotypic analysis shows faster migration, less adhesion, and diminished stress fibers

  • Comparison with wild-type controls reveals actin-remodeling function

• Super-resolution microscopy:

  • Precise localization of CDC14A at actin structures

  • Co-localization with actin regulators like KIBRA at nanoscale resolution

  • Reveals distinct pools at basal bodies with differential functions

• Molecular pathway analysis:

  • CDC14A influences the Hippo pathway through KIBRA

  • KIBRA overexpression rescues phenotypes of CDC14A PD cells

  • Suggests evolutionary conservation of CDC14/Hippo regulatory relationships

• In vitro reconstitution systems:

  • Purified components to study direct effects on actin polymerization

  • Identification of actin-stabilizing function through dephosphorylation of regulators

These techniques have established that CDC14A stabilizes actin filaments, potentially by regulating GEFs and GAPs that control small GTPases .

How can researchers effectively validate CDC14A antibody specificity in complex experimental systems?

Comprehensive validation of CDC14A antibody specificity requires multiple approaches:

• Genetic knockout controls:

  • Use CRISPR/Cas9-generated CDC14A knockout cell lines

  • Example: hTERT-RPE1 CDC14A KO cells with exon 2 removal causing frameshift

  • Antibody signals should disappear in knockout cells

• siRNA knockdown validation:

  • Use CDC14A-specific siRNAs (validated by RT-PCR)

  • Example validation approach from source :

    • Design multiple siRNA duplexes targeting different regions

    • Validate specificity by RT-PCR checking both CDC14A and CDC14B

    • Create rescue constructs with silent mutations in siRNA-targeted regions

    • Demonstrate rescue of phenotype after siRNA + rescue construct

• Recombinant protein controls:

  • Express tagged CDC14A for antibody validation

  • Example: Observe overlap between myc and CDC14A signals in cells overexpressing myc-CDC14A

• Cross-species reactivity testing:

  • Test antibodies across human, mouse, and rat samples

  • Document specific band patterns and molecular weights (66-70 kDa)

• Multiple application validation:

  • Test antibodies in Western blot, immunofluorescence, and immunoprecipitation

  • Note application-specific limitations (some antibodies work in IF but not WB)

• Cross-antibody correlation:

  • Compare results from different antibodies targeting distinct CDC14A epitopes

  • Document concordant and discordant findings

These validation approaches ensure reliable research outcomes and prevent misinterpretation due to antibody artifacts.