KIF15 Antibody

What is KIF15 and what cellular functions does it perform?

KIF15 is a plus-end directed kinesin-like motor enzyme involved in mitotic spindle dynamics and chromosome alignment, making it a key player in cell division processes . It functions as a molecular motor that travels along microtubules during cell division to help establish and maintain the bipolar spindle structure necessary for proper chromosome segregation . KIF15 has also been identified as having partial functional redundancy with KIF11, another mitotic kinesin, as KIF15 can replace essential functions of KIF11 in the creation of the bipolar spindle . This functional redundancy has significant implications for research targeting either protein, as cells may compensate for the loss of one through upregulation of the other.

What applications are KIF15 antibodies commonly used for in research?

KIF15 antibodies are valuable tools for multiple research applications including:

• Western blot analysis to detect and quantify KIF15 protein expression

• Immunohistochemistry to visualize KIF15 in tissue sections, as demonstrated in studies examining KIF15 expression in glioblastoma and prostate cancer tissues

• Immunofluorescence for subcellular localization of KIF15, as shown in HeLa cells using specific antibodies like PACO57476

• ELISA for quantitative detection of KIF15 in various sample types

These applications enable researchers to investigate KIF15 expression patterns, subcellular localization, and potential involvement in normal cellular processes and disease states.

What are the recommended techniques for validating KIF15 antibody specificity?

To ensure the reliability of KIF15 antibody-based experiments, researchers should validate antibody specificity through multiple approaches:

• Genetic controls: Using KIF15 knockdown or knockout cells as negative controls. For example, creating a KIF15 KO cell line via CRISPR/Cas9 technique as described in studies with S462 cells provides an excellent specificity control .

• Peptide competition assays: Pre-incubating the antibody with the immunogen peptide to confirm signal elimination.

• Multiple antibody validation: Using different antibodies targeting distinct KIF15 epitopes to confirm consistent staining patterns.

• Western blot analysis: Confirming a single band of appropriate molecular weight (~160 kDa for human KIF15).

• Cross-reactivity testing: Evaluating the antibody in multiple species to confirm expected reactivity patterns, as illustrated by the PACO57476 antibody which specifically detects human KIF15 .

Thorough validation is essential for obtaining reliable research data, especially when characterizing KIF15's role in complex biological processes like cancer development.

How should researchers optimize protocols for KIF15 immunohistochemistry in different tissue contexts?

Optimizing KIF15 immunohistochemistry protocols requires consideration of tissue-specific factors:

For paraffin-embedded tissues, the following protocol has been successfully employed:

• Deparaffinization of tissue sections

• Antigen retrieval using citrate buffer

• Blocking with 3% H₂O₂ for 10 minutes at room temperature

• Overnight incubation with primary KIF15 antibody at 4°C (typically at 1:50 dilution for commercial antibodies)

• Incubation with HRP-conjugated secondary antibody (e.g., goat anti-rabbit IgG H&L at 1:400) at 37°C for 1 hour

• Visualization using appropriate detection systems and counterstaining

For cultured cells, such as in immunofluorescence applications, protocols typically involve:

• Fixation in 4% formaldehyde

• Permeabilization with 0.2% Triton X-100

• Blocking in 10% normal goat serum

• Overnight incubation with primary antibody at 4°C (recommended dilutions for PACO57476: 1:200-1:500)

• Incubation with fluorophore-conjugated secondary antibody (e.g., Alexa Fluor 488-conjugated anti-rabbit IgG)

• Counterstaining with DAPI for nuclear visualization

Optimization requires testing different antibody concentrations, incubation times, and antigen retrieval methods for each specific tissue type and fixation method.

What methodological approaches can distinguish between KIF15 expression and activity levels?

Distinguishing between KIF15 expression and its functional activity requires complementary approaches:

For expression analysis:

• Western blotting and immunohistochemistry quantify protein levels

• RT-qPCR measures mRNA expression (shown to achieve ~80% reduction in KIF15 expression following siRNA treatment in cell lines like ST88-14)

• Flow cytometry can analyze expression at the single-cell level

For activity assessment:

• Functional assays evaluating mitotic spindle formation and integrity

• Cell viability and proliferation assays following KIF15 modulation (as demonstrated in MPNST cell lines)

• Cell cycle analysis by flow cytometry to detect G2/M arrest following KIF15 inhibition

• Colony formation assays to assess long-term effects of KIF15 activity modulation

• In vivo tumor growth experiments using xenograft models with modified KIF15 expression or activity

Combining these approaches provides a comprehensive understanding of both KIF15 expression and its functional significance in experimental systems.

How does KIF15 knockdown affect cancer cell behavior and what methods are most effective?

KIF15 knockdown has demonstrated significant effects on cancer cell behavior across multiple studies:

In glioma cells, silencing of KIF15 inhibited cell proliferation and stemness, arrested cells in the G2 phase, and induced cell apoptosis . The inhibitory effect was also verified in vivo using xenograft models .

In MPNST cells, RNAi-mediated suppression of KIF15 showed cell line-dependent effects:

• In ST88-14 cells, KIF15 knockdown significantly inhibited cell proliferation

• In S462 cells, with less efficient KIF15 depletion (~60% reduction vs. ~80% in ST88-14), the effects were less pronounced

Methods for effective KIF15 knockdown include:

• siRNA transfection: Using siRNA pools targeting KIF15, achieving 60-80% expression reduction in different cell lines

• CRISPR/Cas9 gene editing: Creating complete KIF15 knockout cell lines for more definitive functional studies

• Chemical inhibitors: Compounds like KIF15-IN-1 have been reported to suppress cancer cell growth

The choice of knockdown method depends on the research question, with transient siRNA approaches suitable for initial screening and CRISPR/Cas9 knockout preferred for in-depth mechanistic studies requiring complete protein elimination.

What molecular mechanisms underlie KIF15's role in cancer progression?

The molecular mechanisms underlying KIF15's role in cancer progression involve multiple pathways:

• Cell cycle regulation: KIF15 knockdown arrests cancer cells in the G2/M phase, indicating its critical role in mitotic progression . This is likely due to disruption of mitotic spindle assembly and function.

• Apoptosis regulation: KIF15 knockdown induces apoptosis in cancer cells, with Human Apoptosis Antibody Array showing upregulation of pro-apoptotic factors including CD40L, cytoC, DR6, and p21, while anti-apoptotic factors like IGF-I and Survivin were downregulated .

• Cell stemness: KIF15 appears to regulate cancer stem cell-like properties, as its knockdown reduces stemness in glioma cells .

• Compensatory mechanisms: KIF15 can functionally compensate for KIF11 inhibition in mitotic spindle formation, which has implications for therapeutic strategies targeting either kinesin .

• Cell viability pathways: KIF15 deficiency sensitizes cancer cells to other therapeutic agents, as demonstrated in S462 cells where KIF15 knockout enhanced sensitivity to KIF11 inhibitors and combined KIF11/BRD4 inhibition .

These mechanisms collectively contribute to KIF15's role as a tumor promoter, making it an attractive therapeutic target in multiple cancer types.

How can researchers effectively target KIF15 in combination with other mitotic kinesins for cancer therapy?

Research indicates that targeting KIF15 in combination with other mitotic kinesins and cancer-related proteins offers promising therapeutic strategies:

• KIF11 and KIF15 dual targeting: Studies have demonstrated that KIF15-deficient cells show increased sensitivity to KIF11 inhibitors like ispinesib and ARRY-520 . This approach exploits the partial functional redundancy between these kinesins, where KIF15 loss prevents compensatory mechanisms that might otherwise confer resistance to KIF11 inhibition.

• Triple inhibition approaches: The combination of KIF11, KIF15, and BRD4 inhibition has shown synergistic antitumoral effects in MPNST cell lines . ARRY-520 (KIF11 inhibitor) combined with JQ1 (BRD4 inhibitor) was particularly effective in KIF15-deficient cells, where it:

  • Induced significant G2/M cell cycle arrest

  • Promoted apoptotic-mediated cell death

  • Drastically reduced colony formation capacity

• Sequential targeting: Researchers might consider sequential inhibition approaches, first depleting KIF15 (via genetic approaches or specific inhibitors) before administering KIF11 inhibitors to maximize efficacy.

• Cell-line specific optimization: Research indicates variable dependence on KIF15 across cancer cell lines, necessitating personalized approaches. For example, ST88-14 cells showed greater sensitivity to KIF15 knockdown than S462 cells .

Researchers should incorporate appropriate controls, including non-tumor cells (like human fibroblasts), to assess potential differential sensitivity between normal and cancer cells to these combination approaches .

What are the optimal dilutions and conditions for using KIF15 antibodies in different applications?

Based on published protocols and manufacturer specifications, the following dilutions and conditions are recommended for KIF15 antibodies:

For immunohistochemistry:

• Primary KIF15 antibody: 1:50 dilution (Fine Test antibody)

• Incubation: Overnight at 4°C

• Secondary antibody: Goat anti-rabbit IgG H&L HRP-conjugated at 1:400 dilution

• Incubation: 37°C for 1 hour

For immunofluorescence:

• Primary KIF15 antibody (e.g., PACO57476): 1:200-1:500 dilution

• Cell preparation: Fixation in 4% formaldehyde, permeabilization with 0.2% Triton X-100, blocking in 10% normal goat serum

• Incubation: Overnight at 4°C

• Secondary antibody: Alexa Fluor 488-conjugated anti-rabbit IgG

For ELISA:

• KIF15 antibody (e.g., PACO57476): 1:2000-1:10000 dilution

These recommendations serve as starting points, and researchers should optimize conditions for their specific experimental systems, antibody lots, and sample types through dilution series and positive/negative controls.

How should researchers design and interpret KIF15 knockdown experiments?

Designing and interpreting KIF15 knockdown experiments requires careful consideration of several factors:

Experimental design recommendations:

• Include multiple knockdown approaches:

  • siRNA pools for transient knockdown (shown to achieve 60-80% reduction in KIF15 expression)

  • CRISPR/Cas9 for complete knockout where appropriate

  • Chemical inhibitors like KIF15-IN-1 as complementary approaches

• Validate knockdown efficiency:

  • Quantify knockdown at both mRNA (RT-qPCR) and protein levels (Western blot)

  • Establish the time course of knockdown effectiveness

• Include comprehensive functional readouts:

  • Cell viability assays (e.g., MTT)

  • Cell proliferation assays

  • Cell cycle analysis by flow cytometry

  • Apoptosis assays

  • Colony formation capacity

  • In vivo tumor growth in xenograft models where feasible

• Essential controls:

  • Non-targeting control siRNA

  • Wild-type parental cell lines for comparison with knockout lines

  • Multiple cancer cell lines to assess generalizability

  • Non-cancerous cells (e.g., fibroblasts) to evaluate cancer specificity

Interpretation considerations:

• Cell line-dependent effects: Different cell lines may show variable sensitivity to KIF15 depletion (e.g., ST88-14 vs. S462)

• Partial vs. complete knockdown: The degree of knockdown achieved may significantly impact phenotypic outcomes

• Compensatory mechanisms: Consider potential upregulation of functionally redundant proteins (e.g., KIF11)

• Time-dependent effects: Distinguish between immediate and long-term consequences of KIF15 depletion

By following these design principles and interpretation guidelines, researchers can generate more reliable and translatable data on KIF15 function in cancer and other contexts.

What techniques are most effective for studying the relationship between KIF15 and other mitotic kinesins?

To investigate the functional relationship between KIF15 and other mitotic kinesins, particularly KIF11, researchers can employ several effective techniques:

• Sequential and simultaneous knockdown/inhibition:

  • Compare the effects of individual kinesin knockdown to combined approaches

  • Study KIF11 inhibition in KIF15-deficient backgrounds to assess functional redundancy

  • Evaluate potential synergistic effects with other targets (e.g., BRD4)

• Sensitivity assays in genetically modified backgrounds:

  • Test KIF11 inhibitors (e.g., ispinesib, ARRY-520) in KIF15 knockout cells

  • Construct dose-response curves to quantify differential sensitivity

  • Calculate synergy scores for drug combinations in different genetic backgrounds

• Rescue experiments:

  • Determine if KIF15 overexpression can rescue phenotypes caused by KIF11 inhibition

  • Identify the minimal domains of KIF15 required for functional compensation

• Proximity-based interaction studies:

  • Proximity ligation assays or FRET to detect potential physical interactions

  • Co-immunoprecipitation to identify binding partners shared between KIF15 and other kinesins

• Imaging approaches:

  • Live-cell imaging to visualize mitotic spindle dynamics in cells with various kinesin modifications

  • Super-resolution microscopy to examine co-localization patterns

• Protein expression analysis:

  • Assess whether inhibition of one kinesin leads to compensatory upregulation of others

  • Study the relationship between expression levels and functional outcomes

These approaches have revealed important findings, such as KIF15's ability to replace essential functions of KIF11 in bipolar spindle formation and the enhanced sensitivity of KIF15-deficient cells to KIF11 inhibitors , highlighting the complex interplay between these motor proteins in mitotic regulation.

How should researchers quantify and interpret KIF15 expression data in patient samples?

Quantification and interpretation of KIF15 expression in patient samples requires standardized approaches:

For immunohistochemistry quantification:

• Score system development: Researchers commonly use semi-quantitative scoring methods that incorporate both staining intensity and percentage of positive cells.

• Cut-off determination: The median of half-quantified expression scores from all samples can serve as a cut-off point to distinguish high versus low KIF15 expression cases .

• Blinding procedures: Pathologists should evaluate slides in a blinded manner to minimize bias.

• Statistical analysis: Apply appropriate statistical tests to compare expression levels between normal and tumor tissues or across different disease stages.

For clinical correlation analysis:

• Use Mann-Whitney U-analysis to evaluate associations between KIF15 expression and clinicopathological parameters like tumor invasion depth .

• Apply Spearman rank correlation analysis to confirm significant correlations .

• Perform survival analysis using Kaplan-Meier curves and log-rank tests to assess relationships between KIF15 expression and patient outcomes .

• Utilize multivariate analyses to determine whether KIF15 expression is an independent prognostic factor.

When interpreting results:

• Consider tissue heterogeneity and the need for multiple sampling within tumors.

• Acknowledge potential differences between protein and mRNA expression patterns.

• Compare findings with publicly available datasets (e.g., TCGA) to validate observations across larger cohorts .

• Interpret expression data in the context of other molecular markers and clinical parameters.

These approaches have revealed significant correlations between high KIF15 expression and advanced pathological features in multiple cancer types, supporting its potential value as a prognostic biomarker .

What are the most informative functional assays for evaluating KIF15 inhibition effects?

When evaluating the effects of KIF15 inhibition, several complementary functional assays provide comprehensive insights:

These assays collectively provide a comprehensive understanding of how KIF15 inhibition affects cancer cell biology across multiple functional domains.