IFT80 Antibody

What is IFT80 and what cellular functions does it perform?

IFT80 is a critical component of the intraflagellar transport (IFT) complex B, which plays an essential role in the development and maintenance of both motile and sensory cilia. It contributes to the assembly and proper positioning of ciliary components through the intraflagellar transport system . IFT80 is also known as WDR56 (WD repeat-containing protein 56) and encoded by the gene IFT80 (intraflagellar transport 80 homolog). The protein has a calculated molecular weight of approximately 88 kDa . At the cellular level, IFT80 functions to transport cargo proteins along the ciliary axoneme, which is crucial for ciliogenesis and ciliary signaling pathways. This transport mechanism is essential for numerous developmental processes and cellular functions that rely on proper cilia formation.

What applications are commonly supported by commercial IFT80 antibodies?

IFT80 antibodies support multiple experimental applications with varying degrees of optimization:

When selecting an antibody, researchers should verify specific reactivity with their target species. For example, Proteintech's antibody (25230-1-AP) has confirmed reactivity with human and mouse samples, while Abnova's antibody (PAB15842) is specifically validated for mouse samples .

What sample preparation methods are optimal for IFT80 detection?

For Western blot applications, optimal sample preparation depends on the cellular localization of IFT80. Since IFT80 is predominantly associated with cilia and intracellular transport structures, a lysis buffer containing both detergent and mechanical disruption is recommended. Based on published protocols, cells should be lysed in a buffer containing protease inhibitors to prevent degradation, as IFT80 can be susceptible to proteolytic cleavage.

For tissue samples:

• Homogenize fresh or flash-frozen tissue in RIPA buffer supplemented with protease inhibitors

• Incubate on ice for 30 minutes with occasional vortexing

• Centrifuge at 12,000 g for 20 minutes at 4°C

• Collect supernatant and determine protein concentration

For adherent cell cultures like NIH/3T3 (where IFT80 antibodies show positive results), scrape cells in PBS followed by centrifugation and resuspension in lysis buffer . When processing intervertebral disc tissues, special considerations are needed due to the high proteoglycan content which can interfere with protein extraction .

How can I troubleshoot and optimize Western blot conditions for IFT80 detection?

When optimizing Western blots for IFT80 detection, consider these methodological approaches:

• Sample loading: Load 30-50 μg of total protein per lane as indicated in validated protocols

• Gel percentage: Use 5% SDS-PAGE gels for optimal separation of the 88-90 kDa IFT80 protein

• Transfer conditions: Wet transfer at lower voltage (30V) overnight at 4°C improves transfer efficiency of larger proteins

• Blocking conditions: 5% non-fat milk in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature

• Antibody dilution: Start with manufacturer's recommended dilution (e.g., 1:500 for ab154933 or 1:500-1:2000 for 25230-1-AP)

• Incubation time: Primary antibody incubation overnight at 4°C improves signal quality

• Detection method: Enhanced chemiluminescence (ECL) with extended exposure times may be necessary

If non-specific bands appear, additional blocking with 2% BSA or increasing wash stringency can improve specificity. The predicted molecular weight of IFT80 is 88 kDa, with observed bands typically between 88-90 kDa .

What are the unique considerations when investigating IFT80's role in bone development and healing?

IFT80 plays a critical role in bone development and healing, requiring specialized experimental approaches to investigate its functions. Research has shown that IFT80 is essential for fracture healing through its regulation of TGF-β signaling in chondrocytes . When designing experiments to study IFT80 in bone contexts, consider:

• Model selection: Conditional knockout models using bone/cartilage-specific Cre drivers (Col2a1-Cre for growth plate and Col1a1-Cre for mature bone cells) have been successfully used to study IFT80 function in vivo

• Timepoint selection: For fracture healing studies, early timepoints (days 7-14) are critical as IFT80 expression is most pronounced during the initial healing phases

• Protein extraction: Bone tissues require specialized extraction protocols due to their mineralized nature; demineralization steps may be necessary

• Complementary techniques: Combine IHC/IF with μCT imaging and biomechanical testing for comprehensive analysis

Recent published research demonstrates that deletion of IFT80 in type II collagen-positive cells disrupts growth plate organization, while deletion in type I collagen-positive cells affects outer annulus fibrosus alignment . This suggests cell type-specific functions of IFT80 that should be considered when designing targeted experiments.

How does IFT80 influence osteoclast differentiation and what methodological approaches can investigate this relationship?

IFT80 negatively regulates osteoclast differentiation through a TRAF6-dependent mechanism . To investigate this relationship:

• In vitro osteoclastogenesis assays: Culture bone marrow-derived macrophages (BMMs) with RANKL and M-CSF, with and without IFT80 manipulation

• TRAP staining: Quantify osteoclast formation by TRAP-positive multinucleated cell counting

• Actin ring formation assays: Assess osteoclast function through visualization of actin rings

• Acridine orange assays: Measure acid production capacity of mature osteoclasts

• Gene expression analysis: Monitor osteoclast markers including TRAP, Ctsk, Dc-stamp, and Atp6v0d2

Research has demonstrated that IFT80 deletion using LysM-Cre (for osteoclast precursors) or Ctsk-Cre (for mature osteoclasts) results in:

• Increased osteoclast numbers (4-fold increase in TRAP+ cells)

• Enhanced actin ring formation (3-fold increase)

• Higher acid content production (1.8-fold increase)

• Elevated expression of osteoclast-specific genes

• Significant bone loss (7-fold decrease in bone volume/total volume)

When investigating the mechanism, focus on TRAF6 protein levels and stability, as IFT80 appears to regulate TRAF6 through modulation of Cbl-mediated proteasomal degradation pathways .

What experimental approaches can effectively study IFT80 in relation to ciliopathies and hedgehog signaling?

Given IFT80's critical role in ciliogenesis and ciliary function, specific experimental approaches can elucidate its relationship to ciliopathies and hedgehog signaling:

• Ciliary visualization techniques:

  • Immunofluorescence using antibodies against ciliary markers (acetylated α-tubulin, Arl13b) alongside IFT80

  • Super-resolution microscopy for detailed ciliary substructure examination

  • Live-cell imaging using fluorescently tagged IFT80 to track dynamics

• Hedgehog pathway assessment:

  • Gli1 and Patched1 expression analysis as direct readouts of pathway activity

  • Gli reporter assays (e.g., 8xGliBS-luciferase) for quantitative measurements

  • SHH stimulation experiments with or without IFT80 manipulation

• Disease modeling approaches:

  • CRISPR/Cas9-mediated introduction of patient-specific IFT80 mutations

  • Patient-derived iPSCs differentiated into relevant cell types

  • Conditionally inducible systems to study temporal requirements

Research has shown that loss of IFT80 causes marked decrease in hedgehog signaling components, including Gli1 and Patched1, in intervertebral disc tissues . This indicates that experiments should focus on tissue-specific effects of IFT80 disruption on hedgehog pathway function.

What considerations are important when studying IFT80 in intervertebral disc development?

Intervertebral disc (IVD) development studies require specialized approaches when investigating IFT80 function:

• Tissue-specific considerations:

  • The IVD comprises three distinct regions: nucleus pulposus (NP), annulus fibrosus (inner and outer), and endplates

  • Each region requires different extraction and preparation protocols

  • IFT80 functions differ between regions, necessitating region-specific analysis

• Ex vivo organ culture system:

  • NP tissue organs can be isolated from the spinal column (L1 to caudal 10) and cultured

  • Adenoviral delivery of Cre or control vectors enables manipulation of IFT80 in IFT80-floxed tissues

  • This approach allows for controlled genetic manipulation in a physiologically relevant context

• Phenotypic analysis:

  • Histological assessment of disc architecture using H&E, Safranin O, and Alcian blue stains

  • Immunohistochemical detection of type I and type II collagen distribution

  • Gene expression analysis of chondrogenic markers (Col2a1, Sox9, Aggrecan)

Deletion of IFT80 in type II collagen-expressing cells disrupts IVD structure with disorganized and decreased growth plate, endplate, and inner annulus fibrosus, while deletion in type I collagen-expressing cells affects the outer annulus fibrosus organization . These differential effects emphasize the importance of cell type-specific approaches when studying IFT80 in the IVD.

What controls should be included when working with IFT80 antibodies?

Proper experimental controls are essential for reliable IFT80 antibody-based studies:

• Positive controls:

  • NIH/3T3 cells, mouse brain tissue, and mouse kidney tissue have been validated as positive control samples

  • NT2D1 whole cell lysate has been validated for human samples

• Negative controls:

  • Primary antibody omission controls

  • IFT80 knockout or knockdown samples (when available)

  • Non-ciliated cell lines as functional negative controls

• Loading controls:

  • β-actin or GAPDH for Western blot normalization

  • For ciliary studies, acetylated α-tubulin or other ciliary markers for co-localization

• Specificity controls:

  • Pre-absorption with immunizing peptide

  • Secondary antibody-only controls

  • Isotype-matched irrelevant antibody controls

When validating a new lot of antibody, comparison with previously validated lots using consistent positive control samples is recommended for ensuring consistency across experiments.

How can I quantitatively assess IFT80 expression changes in experimental settings?

Quantitative assessment of IFT80 expression requires rigorous methodological approaches:

• Western blot densitometry:

  • Normalize IFT80 signal to loading controls

  • Use calibration curves with recombinant standards for absolute quantification

  • Employ technical replicates and biological replicates (minimum n=3)

• qRT-PCR for transcript levels:

  • Design primers spanning exon-exon junctions

  • Validate primer efficiency using standard curves

  • Normalize to multiple reference genes for accuracy

  • Consider using digital PCR for absolute quantification

• Image analysis for immunofluorescence/IHC:

  • Apply consistent threshold settings across experimental groups

  • Measure staining intensity, area, and co-localization parameters

  • Use automated analysis algorithms to reduce bias

  • Report both signal intensity and percentage of positive cells

• Flow cytometry:

  • Quantify IFT80 expression at the single-cell level

  • Assess population heterogeneity in expression levels

  • Gate on appropriate cell populations

For IFT80 knockdown validation, a combination of mRNA and protein quantification is recommended, as post-transcriptional regulatory mechanisms may affect protein levels independently of transcript changes.

What novel research areas are emerging regarding IFT80 beyond its canonical ciliary functions?

Recent research is expanding our understanding of IFT80 beyond its classic role in ciliogenesis:

• Signaling pathway integration:

  • IFT80 negatively regulates osteoclast differentiation through TRAF6-dependent mechanisms

  • Potential involvement in non-canonical Wnt signaling pathways

  • Cross-talk with TGF-β signaling during bone healing

• Cell type-specific functions:

  • Differential roles in type I versus type II collagen-expressing cells

  • Cell-autonomous versus non-cell-autonomous effects on tissue development

  • Lineage-specific requirements during differentiation processes

• Therapeutic targeting potential:

  • IFT80 manipulation as a strategy to enhance bone healing

  • Ciliary-targeted approaches for treating ciliopathies caused by IFT80 mutations

  • Small molecule modulators of IFT80 function or stability

Experimental approaches to explore these emerging areas should combine traditional biochemical methods with advanced imaging techniques, single-cell analysis, and in vivo functional studies with tissue-specific genetic manipulations.

How can I design experiments to investigate potential IFT80 interacting partners?

To identify and characterize IFT80 protein-protein interactions:

• Co-immunoprecipitation approaches:

  • Use IFT80 antibodies to pull down protein complexes

  • Validate interactions with reciprocal IP experiments

  • Consider crosslinking to stabilize transient interactions

• Proximity labeling methods:

  • BioID or TurboID fusion proteins to identify proximal proteins

  • APEX2-based approaches for temporal control

  • Consider ciliary-targeted constructs for organelle-specific interactome mapping

• Mass spectrometry-based approaches:

  • Quantitative proteomics comparing wild-type versus IFT80-deficient samples

  • SILAC or TMT labeling for more precise quantification

  • Focused analysis of ciliary fractions to enrich for relevant interactors

• Yeast two-hybrid screening:

  • Use domain-specific baits to identify direct binding partners

  • Validate hits with biochemical approaches

  • Consider membrane yeast two-hybrid for transmembrane interactors

• Fluorescence-based interaction detection:

  • FRET/FLIM to detect protein-protein interactions in live cells

  • BiFC to visualize interaction events in specific subcellular compartments

  • FCCS to measure interaction kinetics in living cells

Current research suggests potential interactions with both IFT complex components and signaling molecules like TRAF6 and Cbl proteins, which could be promising starting points for interaction studies .