KIF14 Antibody

What is KIF14 and what cellular functions does it regulate?

KIF14 (Kinesin Family Member 14) is a microtubule motor protein that plays crucial roles in multiple cellular processes. It binds to microtubules with high affinity through tubulin heterodimers and possesses ATPase activity. KIF14 regulates several essential biological functions, including:

• Cell division and cytokinesis

• Axonal transport in neuronal cells

• Cell proliferation and apoptosis

• Cell migration and focal adhesion dynamics

• Primary cilium formation and ciliogenesis

During cytokinesis, KIF14 targets to the central spindle and midbody through interactions with PRC1 and citron kinase (CIT) . KIF14 also regulates cell growth through cell cycle progression control, particularly through SCF-dependent proteasomal ubiquitin-dependent protein catabolic processes that control CDKN1B degradation, leading to positive regulation of cyclins including CCNE1, CCND1, and CCNB1 .

What types of KIF14 antibodies are available for research applications?

Several types of KIF14 antibodies are commercially available with varying properties suitable for different experimental applications:

When selecting a KIF14 antibody, researchers should consider the specific application, target species, and whether conjugated antibodies might be beneficial for their experimental design .

How should KIF14 antibodies be validated for experimental use?

Proper validation of KIF14 antibodies is critical to ensure experimental rigor. A comprehensive validation approach includes:

• Specificity testing: Perform knockdown experiments using siRNA or shRNA targeting KIF14 to confirm antibody specificity. For example, studies have validated KIF14 mRNA FISH probes using MDA-MB-231 cells with knocked-down KIF14 compared to parental cells .

• Cross-reactivity assessment: Test the antibody across multiple cell lines or tissues to verify consistent detection patterns. The KIF14 E-3 antibody has been validated for use with mouse, rat, and human samples .

• Multiple detection methods: Validate using orthogonal techniques such as:

  • Western blotting to confirm molecular weight (186 kDa)

  • Immunofluorescence to verify subcellular localization (primarily cytoplasmic with enrichment at microtubule structures during mitosis)

  • Immunoprecipitation followed by mass spectrometry to confirm target identity

• Positive and negative controls: Include appropriate controls in each experiment:

  • Positive controls: Cell lines known to express KIF14 (T-47D, HEK-293T, HeLa cells)

  • Negative controls: KIF14-depleted cells or tissues lacking KIF14 expression

• Dilution optimization: Determine optimal working concentrations for each application:

  • Western Blot: 1:200-1:1000

  • IP: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

  • IHC: 1:50-1:500

What are the optimal conditions for using KIF14 antibodies in immunofluorescence studies?

For optimal immunofluorescence detection of KIF14:

• Cell preparation:

  • Fix cells using 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.2% Triton X-100 in PBS for 5 minutes

  • Block with 3% BSA in PBS for 1 hour at room temperature

• Antibody incubation:

  • Primary antibody: Dilute KIF14 antibody 1:50-1:200 in blocking buffer

  • Incubate overnight at 4°C in a humidified chamber

  • Secondary antibody: Use species-appropriate fluorophore-conjugated antibody at 1:500 dilution

• Co-staining recommendations:

  • For mitotic studies: Co-stain with α-tubulin to visualize microtubules

  • For cytokinesis studies: Co-stain with citron kinase

  • For cilia studies: Co-stain with ARL13B or acetylated tubulin

• Imaging parameters:

  • Use confocal microscopy for high-resolution imaging of KIF14 localization

  • For time-lapse imaging of KIF14 dynamics during cell division, use GFP-tagged KIF14 constructs

• Signal interpretation:

  • In interphase cells: Primarily cytoplasmic with microtubule association

  • During mitosis: Enriched at the central spindle

  • During cytokinesis: Concentrated at the midbody

How can KIF14 antibodies be used to investigate chemotherapy resistance in triple-negative breast cancer?

KIF14 has been implicated in chemotherapy resistance in triple-negative breast cancer (TNBC). A methodological approach to investigating this phenomenon includes:

• Expression correlation analysis:

  • Use KIF14 antibodies for immunohistochemistry on TNBC patient samples before and after neoadjuvant chemotherapy

  • Score KIF14 expression levels and correlate with treatment response

  • Studies have shown that increased KIF14 expression correlates with resistance to neoadjuvant chemotherapy in locally advanced TNBC

• Functional studies using KIF14 modulation:

  • Establish KIF14 knockdown cell lines using siRNA or shRNA

  • Perform KIF14 overexpression in non-cancerous mammary epithelial cells

  • Test differential chemosensitivity using cell viability assays

  • Research has demonstrated that experimental decrease in KIF14 expression increases docetaxel chemosensitivity in TNBC

• Mechanism investigation:

  • Examine AKT phosphorylation status after KIF14 knockdown using phospho-specific antibodies

  • Perform co-immunoprecipitation assays to identify KIF14 interaction partners

  • Rescue experiments using constitutively active AKT1 (myristoylated AKT1) to reverse chemosensitization

  • Evidence indicates that KIF14 knockdown correlates with decreased AKT phosphorylation and activity

• KIF14 inhibitor studies:

  • Test small molecule inhibitors of KIF14 (e.g., (E)-2-(4-isopropyl-3-nitrobenzylidene)hydrazinecarbothioamide)

  • Compare effects with AKT inhibitors like MK-2206

  • Measure chemosensitization effects when combined with standard chemotherapeutics

  • KIF14 inhibition has shown comparable chemosensitization to AKT inhibition when given with docetaxel

What is the role of KIF14 in ciliogenesis and how can antibodies help elucidate this function?

KIF14 plays a critical role in cilia formation. Research methodologies to investigate this function include:

• Depletion studies:

  • Perform KIF14 knockdown using siRNA in ciliated cell models (e.g., hTERT RPE-1 cells)

  • Measure primary cilium formation and length using markers such as ARL13B or acetylated tubulin

  • Studies have shown KIF14 depletion leads to approximately 50% shorter primary cilia compared to controls

• Time-lapse microscopy:

  • Use GFP-ARL13B expressing cell lines to track cilia growth over time

  • Compare cilia dynamics in control versus KIF14-depleted cells

  • Research has demonstrated that differences in cilia length become more pronounced at later time points following KIF14 knockdown

• Basal body component analysis:

  • Examine localization and abundance of basal body components (TTBK2, CP110, CEP164) after KIF14 depletion

  • Use immunofluorescence with appropriate antibodies and quantify signal intensity

  • Studies suggest KIF14 does not affect these early components of ciliogenesis

• Rescue experiments:

  • Establish stable cell lines expressing siRNA-resistant KIF14 constructs

  • Perform knockdown and rescue experiments to confirm specificity of ciliogenesis defects

  • Researchers have used GFP-KIF14siRNA2res expressing hTERT RPE-1 cells for this purpose

• Hedgehog signaling assessment:

  • As cilia are essential for Hedgehog signaling, measure pathway activity using reporter assays

  • Compare Hedgehog target gene expression in control versus KIF14-depleted cells

  • KIF14 loss leads to Hedgehog signaling defects

How can researchers address non-specific binding issues with KIF14 antibodies?

Non-specific binding is a common challenge with antibodies. For KIF14 antibodies, consider these methodological approaches:

• Optimization of blocking conditions:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Extend blocking time to 2 hours at room temperature

  • Include 0.1-0.3% Triton X-100 in blocking solution to reduce non-specific hydrophobic interactions

• Antibody dilution optimization:

  • Perform titration experiments (e.g., 1:50, 1:100, 1:200, 1:500, 1:1000)

  • Determine optimal signal-to-noise ratio for each application

  • For Western blot applications with KIF14 antibodies, dilutions between 1:200-1:1000 are recommended

• Sample preparation considerations:

  • For tissue samples: Optimize antigen retrieval methods (test both citrate buffer pH 6.0 and TE buffer pH 9.0)

  • For cell lines: Use appropriate fixation methods (4% PFA for immunofluorescence, ice-cold methanol for specific epitopes)

• Validation with multiple antibodies:

  • Test different KIF14 antibodies targeting distinct epitopes

  • Compare staining patterns for consistency

  • Four commercially available KIF14 antibodies have been tested: No. A300-912A (Bethyl), No. ABT46 (Millipore), and No. 48562 and No. 365553 (Santa Cruz)

• Controls to include:

  • Peptide competition assays to confirm binding specificity

  • Antibody omission controls to assess secondary antibody specificity

  • KIF14 knockdown samples as negative controls

What are the considerations for using KIF14 antibodies in tissue microarrays for cancer research?

When using KIF14 antibodies in tissue microarray (TMA) analyses for cancer research:

• Antibody selection and validation:

  • Choose antibodies validated for immunohistochemistry in paraffin-embedded tissues

  • Confirm specificity in positive and negative control tissues

  • The rabbit polyclonal antibody (ab71155) has been validated for IHC-P applications

• Staining protocol optimization:

  • For KIF14 IHC, use antigen retrieval with TE buffer pH 9.0 or alternatively citrate buffer pH 6.0

  • Optimize primary antibody dilution (typically 1:50-1:500)

  • Use appropriate detection systems (HRP/DAB or fluorescent-based)

• Scoring methodology:

  • Develop a consistent scoring system for KIF14 expression (e.g., H-score, Allred score)

  • Assess both staining intensity and percentage of positive cells

  • Consider automated image analysis for objective quantification

• Clinical data correlation:

  • Collect comprehensive patient data including treatment history and outcomes

  • Correlate KIF14 expression with clinicopathological parameters

  • Research has demonstrated that high KIF14 expression in primary tumors correlates with chemoresistance

• Multi-marker analysis:

  • Consider co-staining for KIF14 and AKT pathway components

  • Perform multiplexed immunofluorescence to assess co-localization patterns

  • Analyze correlations between KIF14 and other potential biomarkers

How can KIF14 antibodies be used to investigate the role of KIF14 in neuronal development?

KIF14 plays important roles in neuronal development. Methodological approaches include:

• Expression analysis in developing neural tissues:

  • Use KIF14 antibodies for immunohistochemistry on brain sections at different developmental stages

  • Perform Western blot analysis to quantify expression levels across development

  • Correlate with markers of neurogenesis and neural differentiation

• Primary neural cell culture studies:

  • Isolate primary neurons from embryonic or postnatal brains

  • Manipulate KIF14 expression using siRNA or overexpression constructs

  • Analyze effects on neurite outgrowth, branching, and synaptic development

  • Use immunofluorescence with KIF14 antibodies to track subcellular localization

• Investigation of KIF14's role in axonal transport:

  • Perform live imaging of fluorescently-tagged cargo in KIF14-depleted neurons

  • Conduct co-immunoprecipitation studies to identify neuronal KIF14 cargo

  • Use super-resolution microscopy to visualize KIF14-cargo complexes along axonal microtubules

• Developmental phenotyping:

  • During late neurogenesis, KIF14 regulates cerebellar, cerebral cortex, and olfactory bulb development

  • Use KIF14 antibodies to examine expression patterns in specific brain regions

  • Correlate KIF14 expression with apoptosis markers, proliferation markers, and cell division indices

What methodological approaches can be used to investigate KIF14-mediated regulation of AKT phosphorylation?

To investigate the mechanism by which KIF14 regulates AKT phosphorylation:

• Co-localization studies:

  • Perform dual immunofluorescence for KIF14 and phospho-AKT

  • Use live-cell imaging with fluorescently tagged proteins

  • Research has confirmed an insulin-induced temporal co-localization of KIF14 and AKT at the plasma membrane

• Biochemical interaction analyses:

  • Conduct co-immunoprecipitation assays using KIF14 antibodies

  • Perform proximity ligation assays to detect direct interactions in situ

  • Use pull-down assays with recombinant proteins to map interaction domains

• Functional studies:

  • Modulate KIF14 expression/activity and measure AKT phosphorylation at specific residues (Ser473, Thr308)

  • Use phospho-specific antibodies in Western blotting

  • Compare effects on AKT substrates to confirm pathway activation/inhibition

• Inhibitor studies:

  • Test KIF14 inhibitors ((E)-2-(4-isopropyl-3-nitrobenzylidene)hydrazinecarbothioamide) on AKT phosphorylation

  • Compare with direct AKT inhibitors like MK-2206

  • KIF14 inhibition shows a concentration-dependent effect on AKT phosphorylation and cleaved caspase 3 levels

• Pathway dissection:

  • Investigate the effects of KIF14 modulation on upstream AKT regulators (PI3K, PDK1, mTORC2)

  • Determine if KIF14 affects AKT phosphatase activity (PP2A, PHLPP)

  • Use rescue experiments with constitutively active AKT to bypass KIF14 regulation