KIFC3 Antibody

What is KIFC3 and what cellular functions does it regulate?

KIFC3 is a C-terminal motor domain-type kinesin superfamily protein that functions as a minus end-directed microtubule motor. In polarized epithelial cells, KIFC3 localizes on membrane organelles immediately beneath the apical plasma membrane of renal tubular epithelial cells in vivo and polarized MDCK II cells in vitro . Its primary function involves the transport of membrane vesicles along microtubules, particularly in apically targeted transport pathways within polarized cells . Additionally, KIFC3 plays crucial roles in Golgi positioning and integration, as disruption of the kifC3 gene can induce fragmentation of the Golgi apparatus under conditions such as cholesterol depletion . Recent research has also highlighted KIFC3's involvement in cancer progression, particularly in non-small cell lung cancer (NSCLC) .

What applications can KIFC3 antibodies be used for in research?

KIFC3 antibodies serve multiple research applications across various technical platforms. According to validated protocols, KIFC3 antibodies have demonstrated effectiveness in:

• Western Blot (WB): Detects KIFC3 protein at 80-85 kDa and 95-100 kDa molecular weights with recommended dilutions of 1:500-1:1000 .

• Immunohistochemistry (IHC): Effectively labels KIFC3 in tissue sections at dilutions of 1:50-1:500, with positive detection in human intrahepatic cholangiocarcinoma tissue .

• Immunofluorescence (IF): Visualizes KIFC3 localization in cellular compartments .

• Immunoprecipitation (IP): Successfully pulls down KIFC3 using 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate .

• Co-immunoprecipitation (CoIP): Detects KIFC3 interactions with other proteins, which has been particularly valuable in studying KIFC3's association with transport vesicles and signaling pathway components .

• ELISA: Quantifies KIFC3 expression levels .

These applications have been validated with human and mouse samples, making KIFC3 antibodies versatile tools for cross-species research .

How should researchers validate KIFC3 antibody specificity?

Validating KIFC3 antibody specificity requires a multi-faceted approach to ensure experimental reliability:

• Knockout/Knockdown Controls: Utilize KIFC3 knockout or knockdown models as negative controls. Published research demonstrates that KIFC3 antibodies show no reactivity in KIFC3 knockout mice, confirming specificity . The antibody recognized multiple protein bands in wild-type kidney samples, but these bands were absent in KIFC3 knockout mice .

• Western Blot Validation: Verify the antibody detects bands at the expected molecular weights (80-85 kDa and 95-100 kDa) . Multiple bands may represent post-translational modifications or NH₂-terminally truncated proteins, as suggested by research showing only the highest band was recognized by anti-KIFC3 NH₂-terminus antibody .

• Cross-reactivity Assessment: Test antibody performance across multiple species (human, mouse) and tissue types to confirm consistent recognition patterns .

• Positive Control Tissues: Include known KIFC3-expressing tissues such as mouse lung, mouse kidney, and human intrahepatic cholangiocarcinoma as positive controls in validation experiments .

• Immunofluorescence Correlation: Compare KIFC3 staining patterns with known interacting partners or subcellular structures (e.g., apical membrane in polarized cells, Golgi apparatus in adrenocortical cells) to confirm expected localization patterns .

How can researchers effectively investigate KIFC3's role in cancer progression?

Investigating KIFC3's role in cancer progression requires sophisticated experimental designs that address multiple aspects of cancer biology:

What methodological approaches should be used to study KIFC3's role in cellular transport mechanisms?

Studying KIFC3's role in cellular transport requires specialized methodologies that capture the dynamic nature of these processes:

• Live Cell Imaging:

  • Track KIFC3-associated vesicle movement using fluorescently tagged KIFC3 constructs.

  • Employ time-lapse confocal microscopy to visualize the kinetics of apically targeted transport in polarized epithelial cells .

• Subcellular Fractionation and Vesicle Isolation:

  • Isolate membrane organelles using flotation assays coupled with detergent extraction to identify KIFC3 association with specific vesicle populations.

  • Research has shown that KIFC3 associates with Triton X-100-insoluble membrane organelles that overlap with apically transported TGN-derived vesicles .

• Transport Assays with Perturbation Approaches:

  • Use low-temperature blocking (18.5°C incubation) to partially inhibit vesicular exit from the TGN and observe KIFC3 redistribution.

  • Apply brefeldin A (BFA) treatment (2 μg/ml) to selectively inhibit TGN to apical surface transport.

  • Quantify changes in apical localization using quantitative confocal microscopy after immunofluorescent staining .

• Protein-Protein Interaction Studies:

  • Conduct co-immunoprecipitation assays to confirm interactions between KIFC3 and transport vesicle components.

  • Perform GST pulldown experiments to identify specific interactions, such as those demonstrated between KIFC3 and annexin XIIIb, a membrane protein characteristic of apically transported vesicles .

• Functional Motor Assays:

  • Express recombinant KIFC3 motor domain constructs to assess ATPase activity in the presence of microtubules.

  • Determine motor polarity and processivity characteristics, which are crucial for understanding KIFC3's role in directed transport .

How can researchers differentiate between KIFC3-dependent and dynein-dependent transport pathways?

Distinguishing between KIFC3-dependent and dynein-dependent transport pathways requires careful experimental design:

• Molecular Composition Analysis:

  • Perform immunoisolation of transport vesicles followed by proteomic analysis to define the complete composition of KIFC3-associated vesicles versus dynein-associated vesicles.

  • Research has demonstrated the absence of the dynein-dynactin complex on KIFC3-associated vesicles, suggesting at least two independent apically targeted transport pathways .

• Selective Inhibition Approaches:

  • Use dominant-negative constructs of KIFC3 to specifically inhibit KIFC3-dependent transport without affecting dynein function.

  • Apply dynein-specific inhibitors or dominant-negative constructs to selectively block dynein-dependent transport.

  • Compare the effects of these selective inhibitions on different cargo molecules to map pathway-specific transport routes.

• Cargo-Specific Transport Assays:

  • Track the transport of specific cargoes known to utilize different motor proteins.

  • Identify distinguishing characteristics of vesicles transported by each pathway, such as size, density, or membrane composition.

• Double Knockdown/Knockout Experiments:

  • Create single and double knockdowns/knockouts of KIFC3 and dynein components.

  • Compare phenotypes to identify synergistic or independent effects that would indicate separate or overlapping pathways.

What considerations are important when designing KIFC3 knockdown experiments?

Designing effective KIFC3 knockdown experiments requires careful consideration of multiple factors:

• Knockdown Method Selection:

  • Small interfering RNA (siRNA): Provides transient knockdown suitable for short-term experiments. This approach has been successfully used to demonstrate that KIFC3 knockdown suppresses proliferation, invasion, and migration in NSCLC cells .

  • Short hairpin RNA (shRNA): Offers more stable, long-term knockdown for extended studies or in vivo experiments.

  • CRISPR-Cas9: Provides complete gene knockout when assessing null phenotypes is preferred.

• Validation of Knockdown Efficiency:

  • Quantify KIFC3 mRNA levels using RT-PCR to confirm transcriptional suppression.

  • Assess protein levels via western blot to verify translational inhibition, using validated KIFC3 antibodies at recommended dilutions (1:500-1:1000) .

  • Include appropriate controls: scrambled siRNA/shRNA sequences or non-targeting CRISPR guides.

• Phenotypic Analysis:

  • Proliferation: Employ MTT and colony formation assays to assess effects on cell growth .

  • Migration: Use wound healing assays to measure migratory capability .

  • Invasion: Conduct Matrigel invasion assays to evaluate invasive potential .

  • Signaling pathway analysis: Measure protein levels of PI3K/Akt pathway components via western blot to determine mechanistic effects .

• Rescue Experiments:

  • Express siRNA-resistant KIFC3 constructs to confirm phenotype specificity.

  • Use mutant KIFC3 constructs (e.g., motor-dead variants) to identify which domains are essential for observed functions.

• Timing Considerations:

  • Account for KIFC3 protein half-life when designing experimental timelines.

  • Allow sufficient time after knockdown before phenotypic assessment, particularly for processes that may have delayed effects.

How should researchers approach studying KIFC3's interaction with the PI3K/Akt signaling pathway?

Investigating KIFC3's interaction with the PI3K/Akt pathway requires multifaceted experimental approaches:

• Protein-Protein Interaction Analysis:

  • Perform co-immunoprecipitation assays to confirm direct interaction between KIFC3 and PI3K components. Research has confirmed interaction between endogenous/exogenous KIFC3 and PI3Kp85α .

  • Use GST pulldown assays to map the specific binding domains mediating these interactions.

  • Apply proximity ligation assays to visualize interactions in intact cells.

• Pathway Activation Assessment:

  • Analyze phosphorylation states of key PI3K/Akt pathway components (p-Akt) following KIFC3 overexpression or knockdown.

  • Quantify levels of downstream effectors (cyclin D1, CDK4, CDK6, RhoA, RhoC, and MMP2) to comprehensively map pathway modulation .

• Pharmacological Intervention:

  • Apply PI3K/Akt pathway inhibitors (e.g., LY294002) to determine if KIFC3's cellular effects are dependent on pathway activation.

  • Research has shown that LY294002 can inhibit the promotional effects of KIFC3 overexpression, confirming pathway dependence .

• Functional Rescue Experiments:

  • Attempt to rescue KIFC3 knockdown phenotypes through constitutive activation of PI3K/Akt pathway components.

  • Design KIFC3 mutants with altered ability to interact with PI3K components to define functional domains.

• In Vivo Validation:

  • Establish xenograft models with KIFC3-manipulated cells to confirm pathway interactions in a physiological context.

  • Combine KIFC3 manipulation with pathway inhibitors in animal models to validate therapeutic potential .

What are the optimal conditions for using KIFC3 antibody in immunohistochemistry applications?

Optimizing KIFC3 antibody use in immunohistochemistry requires careful attention to several technical parameters:

• Antigen Retrieval Method:

  • Primary recommendation: Use TE buffer at pH 9.0 for optimal epitope exposure.

  • Alternative approach: Citrate buffer at pH 6.0 may be used if TE buffer yields suboptimal results .

• Antibody Dilution Range:

  • Recommended dilution: 1:50-1:500 depending on tissue type and detection system.

  • Optimization advice: Perform titration experiments with each new tissue type or detection system .

• Positive Control Selection:

  • Human intrahepatic cholangiocarcinoma tissue has been validated as a reliable positive control for KIFC3 immunostaining .

  • Mouse kidney and lung tissues also show consistent KIFC3 expression and can serve as additional positive controls .

• Detection System Considerations:

  • Use amplification systems for low-abundance expression.

  • Consider chromogen selection based on colocalization needs with other markers.

• Counterstaining Parameters:

  • Adjust hematoxylin timing to ensure nuclear detail without obscuring cytoplasmic KIFC3 staining.

  • Monitor counterstain pH to prevent interference with antibody binding.

How can conflicting data regarding KIFC3 expression patterns be resolved?

When encountering conflicting data about KIFC3 expression or function, researchers should implement a systematic reconciliation approach:

• Antibody Validation Comparison:

  • Compare antibody specificity using western blot on the same samples with multiple antibodies targeting different KIFC3 epitopes.

  • Verify antibody specificity using KIFC3 knockout/knockdown controls across different experimental systems .

• Isoform Analysis:

  • Investigate potential splice variants or post-translational modifications that might explain discrepancies. Research has noted multiple bands in western blots that may represent NH₂-terminally truncated proteins produced by posttranslational modification or specific degradation pathways .

  • Perform RT-PCR with primers targeting different regions to identify possible transcript variants.

• Context-Dependent Expression:

  • Evaluate cell type-specific expression patterns across different tissues and cell lines.

  • Assess whether expression changes under different physiological or experimental conditions.

• Methodological Standardization:

  • Standardize sample preparation, fixation methods, and antibody dilutions across experiments.

  • Document and control variables that might influence detection, such as tissue handling, fixation duration, and antibody lot variations.

• Quantification Approaches:

  • Implement multiple quantification methods (e.g., western blot, qPCR, immunofluorescence intensity) to cross-validate findings.

  • Use appropriate statistical analyses to determine the significance of observed differences.

What are the most effective protocols for co-immunoprecipitation using KIFC3 antibodies?

Optimizing co-immunoprecipitation (Co-IP) experiments with KIFC3 antibodies requires attention to several critical factors:

• Antibody Amount Optimization:

  • Recommended starting range: 0.5-4.0 μg of KIFC3 antibody for 1.0-3.0 mg of total protein lysate .

  • Titration advice: Optimize antibody:lysate ratio for each new cell type or experimental condition.

• Lysis Buffer Selection:

  • For membrane-associated complexes: Use buffers containing mild detergents (e.g., 1% NP-40 or 0.5% Triton X-100) to preserve protein-protein interactions while solubilizing membrane components.

  • For cytoskeletal interactions: Consider specialized buffers that maintain microtubule integrity during extraction.

• Pre-clearing Strategy:

  • Implement thorough pre-clearing with protein A/G beads to reduce non-specific binding.

  • Include appropriate IgG controls matched to the host species of the KIFC3 antibody.

• Interaction Preservation Techniques:

  • Cross-linking: Consider mild cross-linking (0.5-1% formaldehyde) for transient interactions.

  • Temperature control: Maintain samples at 4°C throughout the procedure to preserve complex integrity.

• Elution and Detection Optimization:

  • Mild elution: Use non-denaturing conditions for subsequent enzymatic assays.

  • Complete elution: Apply reducing SDS buffer for comprehensive western blot analysis.

  • Enhanced detection: Consider silver staining or highly sensitive western blot systems for low-abundance interacting partners.

How can KIFC3 research inform therapeutic strategies for NSCLC treatment?

KIFC3's role in NSCLC progression suggests several therapeutic development approaches:

What techniques are emerging for studying KIFC3's role in microtubule organization and dynamics?

Advanced techniques for investigating KIFC3's microtubule functions include:

• Super-Resolution Microscopy:

  • Structured illumination microscopy (SIM) can resolve KIFC3 localization along microtubules with ~100 nm resolution.

  • Single-molecule localization microscopy techniques (STORM/PALM) can achieve even higher resolution (~20 nm) to precisely map KIFC3 distribution relative to microtubule structures.

• Live-Cell Force Measurement:

  • Optical tweezers can measure the force generation capabilities of single KIFC3 motor proteins.

  • Magnetic tweezers allow long-duration force measurements to characterize KIFC3's processivity and load-bearing properties.

• Microtubule Dynamics Visualization:

  • EB1-GFP tracking can reveal how KIFC3 influences microtubule plus-end dynamics.

  • Photoactivatable tubulin can help track microtubule turnover rates in the presence or absence of KIFC3.

• In Vitro Reconstitution Systems:

  • Purified component systems using recombinant KIFC3 and fluorescently labeled microtubules can directly measure motor activity and microtubule bundling capabilities.

  • Microfluidic chambers allow controlled introduction of regulatory factors to assess their effects on KIFC3-microtubule interactions.

• Cryo-Electron Microscopy:

  • High-resolution structural analysis of KIFC3-microtubule complexes can reveal the molecular basis of motor function and regulation.

  • Comparative analysis with other kinesin family members can highlight unique structural features of KIFC3.