OSTF1 Antibody

What is OSTF1 and what cellular functions does it perform?

OSTF1 (also known as OSF, SH3P2) is a 24 kDa protein that induces bone resorption through signaling cascades leading to enhanced osteoclast formation and activity . It functions through multiple protein interactions, notably with F-actin, the non-receptor tyrosine kinase c-Src, and E3 ubiquitin-protein ligase Cbl . The co-localization of OSTF1 with Cbl in osteoclast podosomes is particularly significant for bone-resorption properties . OSTF1 also interacts with Survival of Motor Neuron proteins (SMN1 and SMN2), suggesting potential functions beyond bone metabolism .

Which experimental applications are suitable for OSTF1 antibodies?

OSTF1 antibodies have been validated for multiple applications with specific optimal conditions:

These applications allow comprehensive analysis of OSTF1 expression, localization, and interactions in various experimental contexts .

What is the subcellular localization pattern of OSTF1?

Immunofluorescence studies consistently show OSTF1 predominantly localizes to the cytoplasm . Confocal imaging of HeLa and HepG2 cells labeled with anti-OSTF1 antibodies reveals a distinct cytoplasmic staining pattern without significant nuclear localization . In specialized cells like osteoclasts, OSTF1 co-localizes with Cbl in podosomes, which are adhesion structures important for bone resorption . This specific localization pattern supports OSTF1's functional role in cytoskeletal organization and cellular adhesion processes .

Which tissues and cell types express OSTF1?

OSTF1 exhibits a diverse expression pattern across multiple tissues:

• Neural tissues: Strong expression in brain blood vessels (both large and small), ventricles, choroid plexus, and presumptive oligodendrocytes and their precursors (except in cerebellum)

• Vascular system: Prominent expression in the capillary network of the spinal cord from early developmental stages (E11) through adulthood

• Cell lines: Readily detectable in HeLa (cervical cancer), HepG2 (liver cancer), and MCF7 (breast cancer) cell lines

• Other tissues: Expression detected in human fetal liver, fetal brain, fetal kidney, and tonsil

This broad expression pattern suggests OSTF1 may perform functions beyond its established role in bone metabolism .

What are the advantages of using recombinant monoclonal antibodies for OSTF1 detection?

Recombinant monoclonal antibodies, such as the rabbit monoclonal [EP15457] to OSTF1 (ab202901), offer several significant advantages over traditional antibodies:

• High batch-to-batch consistency and reproducibility: Ensures reliable results across experiments and time

• Improved sensitivity and specificity: Recognizes a single epitope with high affinity, reducing background and cross-reactivity

• Long-term security of supply: Production method ensures consistent availability

• Animal-free production: Ethical advantages and reduced variability compared to animal-derived antibodies

These features are particularly valuable for quantitative applications and longitudinal studies where consistent reagent performance is critical .

How do different OSTF1 antibodies compare in specificity and sensitivity?

Two primary types of OSTF1 antibodies are available with distinct characteristics:

For critical experiments, using both antibody types can provide complementary data. Western blotting shows both antibodies detect the expected 24 kDa band, confirming target specificity .

What methodologies are most effective for studying OSTF1 protein interactions?

Several complementary approaches can be employed to study OSTF1 protein interactions:

• Co-immunoprecipitation (Co-IP): The most direct method for studying endogenous interactions. OSTF1 can be immunoprecipitated using antibodies like ab202901 (1/30 dilution) from MCF7 cells, followed by Western blotting for interaction partners .

• Affinity purification coupled with mass spectrometry: An unbiased approach for identifying novel interaction partners. This has been successfully implemented using V5-tagged OSTF1 expressed in HEK293 cells followed by anti-V5 magnetic agarose pulldown and mass spectrometry analysis .

• Protein-protein binding assays: Techniques like peptide arrays have been used to map specific binding domains within OSTF1 that interact with partners like F-actin, c-Src, and Cbl .

• Yeast two-hybrid screening: Has successfully identified interactions between OSTF1 and partners like SMN1/SMN2 .

• Imaging-based approaches: Immunofluorescence co-localization studies have demonstrated the spatial relationship between OSTF1 and Cbl in osteoclast podosomes .

When studying OSTF1 interactions, it's critical to include appropriate controls, such as isotype control antibodies (e.g., rabbit monoclonal IgG) for immunoprecipitation experiments .

What molecular mechanisms underlie OSTF1's role in bone resorption?

OSTF1 promotes bone resorption through multiple molecular mechanisms:

• Signaling cascade activation: OSTF1 appears to trigger signaling pathways that enhance osteoclast formation and activity, resulting in increased bone resorption .

• c-Src interaction: The binding of OSTF1 to c-Src may modulate this key tyrosine kinase's activity, which is essential for osteoclast function and cytoskeletal organization .

• Podosome regulation: OSTF1 co-localizes with Cbl in podosomes of osteoclast-like cells, potentially regulating these adhesion structures that are critical for bone resorption .

• Cytoskeletal organization: Through its interaction with F-actin, OSTF1 may influence cytoskeletal dynamics necessary for osteoclast attachment to bone surfaces and formation of the sealing zone .

• Regulatory factor secretion: OSTF1 may promote the secretion of factors that enhance osteoclast differentiation and function, though the specific factors remain to be fully characterized .

Future research using OSTF1 knockout models and osteoclast-specific manipulations will further clarify these mechanisms.

What are the optimal conditions for detecting OSTF1 in different experimental applications?

Optimal conditions for OSTF1 detection vary by application:

Western Blotting:

• Antibody dilutions: 1/5000 (ab202901) or 0.04-0.4 μg/mL (HPA020514)

• Blocking buffer: 5% non-fat dry milk in TBST

• Loading controls: β-actin antibodies have been validated

• Positive controls: HeLa, HepG2, MCF7 whole cell lysates; human fetal liver tissue

Immunoprecipitation:

• Antibody: ab202901 at 1/30 dilution for IP, 1/1000 for detection by WB

• Lysis buffer: IP lysis buffer (50 mM Tris pH 7.5, 1% Triton-X100, 150 mM NaCl)

• Input material: 1mg of whole cell lysate from MCF7 cells

Immunofluorescence:

• Fixation: 4% paraformaldehyde

• Permeabilization: 0.1% Triton X-100

• Antibody dilutions: 1/250 (ab202901) or 0.25-2 μg/mL (HPA020514)

• Counterstaining: DAPI for nuclei, anti-tubulin for cytoskeletal context

Immunohistochemistry:

• Antigen retrieval: Heat-mediated with Tris/EDTA buffer pH 9.0

• Antibody dilutions: 1/500 (ab202901) or 1:50-1:200 (HPA020514)

• Detection system: HRP-conjugated secondary antibody with hematoxylin counterstain

How can OSTF1 knockout or knockdown models be utilized to study its function?

OSTF1 knockout models provide powerful tools for understanding its physiological roles:

• Phenotypic characterization: Analysis of bone density, structure, and remodeling in OSTF1 knockout mice can reveal its importance in skeletal homeostasis .

• Cell-autonomous effects: Isolated cells from knockout animals or CRISPR-edited cell lines can be used to study OSTF1's direct role in cellular functions including:

  • Osteoclast differentiation and activity

  • Cell migration (particularly relevant given OSTF1's negative impact on cell motility)

  • Protein interaction networks

• Molecular pathway analysis: Comparing signaling pathway activation between wild-type and OSTF1-deficient cells can identify downstream effectors.

• Rescue experiments: Re-introducing wild-type or mutant OSTF1 into knockout cells can identify critical functional domains.

• Disease models: Crossing OSTF1 knockout mice with disease model strains can reveal its role in pathological conditions like osteoporosis or inflammatory bone loss.

When interpreting results from knockout models, it's important to consider potential compensatory mechanisms that may mask phenotypes .

What is the relationship between OSTF1 expression and cell motility?

Experimental evidence indicates OSTF1 negatively regulates cell motility:

• Overexpression studies: Overexpression of OSTF1 in HeLa cells significantly reduces cell motility in transwell assays, establishing OSTF1 as a negative regulator of migration .

• Cytoskeletal interaction mechanism: OSTF1 directly interacts with F-actin, potentially stabilizing the cytoskeleton and restricting dynamic changes required for cell movement .

• Signaling pathway implications: The interaction between OSTF1 and c-Src may modulate signaling pathways that control cell migration, as c-Src is a key regulator of cell motility .

• Adhesion structure regulation: OSTF1's presence in podosomes suggests it may influence cell-substrate adhesion dynamics, which are critical for coordinated cell movement .

This negative regulation of cell motility has significant implications for understanding OSTF1's role in both normal physiology and pathological conditions like cancer metastasis, where altered cell migration is a hallmark feature.

What approaches are effective for studying post-translational modifications of OSTF1?

Multiple complementary approaches can be used to investigate OSTF1 post-translational modifications:

• Mass spectrometry-based proteomics:

  • Immunoprecipitate OSTF1 using validated antibodies (ab202901 or HPA020514)

  • Perform tryptic digestion and LC-MS/MS analysis

  • Compare observed peptide masses with theoretical masses to identify modifications

  • Use phospho-enrichment techniques for phosphorylation-specific analysis

• Western blotting with modification-specific detection:

  • Use phospho-specific antibodies after OSTF1 immunoprecipitation

  • Employ antibodies against ubiquitin, SUMO, or acetyl-lysine to detect these modifications

  • Compare migration patterns before and after phosphatase treatment

• In vitro modification assays:

  • Incubate purified OSTF1 with candidate modifying enzymes (kinases, E3 ligases)

  • Detect modifications using specific antibodies or radioactive labeling

• Site-directed mutagenesis:

  • Mutate potential modification sites and assess functional consequences

  • Compare wild-type and mutant OSTF1 in functional assays

These approaches can reveal how post-translational modifications regulate OSTF1's activity, localization, stability, and protein interactions.

What methodological challenges exist when studying OSTF1 in primary osteoclast cultures?

Investigating OSTF1 in primary osteoclast cultures presents several technical challenges:

• Culture establishment and maintenance:

  • Osteoclasts require specific growth factors (M-CSF and RANKL) for differentiation

  • Their multinucleated nature and limited lifespan complicate long-term studies

  • Heterogeneity in differentiation state can introduce experimental variability

• OSTF1 detection specificity:

  • Ensuring antibodies specifically recognize OSTF1 among related SH3-domain proteins

  • Need for thorough controls including OSTF1-deficient cells

  • Potential cross-reactivity with other proteins expressed in osteoclasts

• Functional assessment:

  • Correlating molecular changes with functional outcomes (bone resorption)

  • Distinguishing direct OSTF1 effects from indirect effects through interaction partners

  • Need for specialized assays like pit formation on bone or dentine slices

• Genetic manipulation:

  • Primary osteoclasts are challenging to transfect efficiently

  • Short lifespan limits expression time for introduced constructs

  • May require manipulation of precursors before differentiation

• Co-localization studies:

  • OSTF1's co-localization with Cbl in podosomes requires high-resolution imaging

  • Podosomes are dynamic structures sensitive to fixation conditions

  • Need for careful optimization of immunofluorescence protocols

What quantitative methods can accurately measure OSTF1 expression?

Multiple complementary approaches can quantify OSTF1 expression with varying advantages:

• Quantitative Western blotting:

  • Semi-quantitative analysis of total OSTF1 protein levels

  • Requires normalization to validated loading controls (β-actin)

  • Recommended antibody dilutions: 1:10,000 (Bethyl A303-004A) or 1:200 (Atlas HPA020514)

  • Can detect changes in protein size due to modifications

• Flow cytometry:

  • Quantifies OSTF1 at the single-cell level

  • Can correlate OSTF1 expression with other cellular parameters

  • Requires intracellular staining protocol with antibody dilution of 1/100 (ab202901)

  • Allows analysis of expression heterogeneity within populations

• Immunofluorescence quantification:

  • Provides spatial information about OSTF1 expression

  • Can be quantified using image analysis software

  • Recommended antibody dilution of 1/250 (ab202901)

  • Useful for co-localization studies with interaction partners

• qRT-PCR:

  • Measures OSTF1 mRNA levels with high sensitivity

  • Cannot account for post-transcriptional regulation

  • Useful for examining transcriptional regulation mechanisms

  • Requires careful primer design and validation

For all quantification methods, appropriate controls are essential, including positive controls (HeLa, HepG2, or MCF7 cells) and negative controls (ideally OSTF1 knockout/knockdown samples).

What considerations are critical for studying OSTF1 in disease models?

When investigating OSTF1 in disease contexts, several key factors should be considered:

• Disease relevance selection:

  • Bone disorders (osteoporosis, Paget's disease) given OSTF1's role in bone resorption

  • Vascular conditions, based on expression in brain vessels and capillary networks

  • Hepatocellular carcinoma, which shows cytoplasmic OSTF1 expression

  • Potential neurological disorders, given interaction with SMN proteins

• Expression analysis approach:

  • Compare OSTF1 levels between normal and diseased tissues

  • Evaluate both mRNA and protein expression

  • Consider cell-type specific expression in heterogeneous samples

  • Correlate expression with disease severity or progression

• Functional investigation:

  • Assess how disease-relevant stimuli affect OSTF1 expression or function

  • Determine if OSTF1 manipulation (knockdown/overexpression) impacts disease phenotypes

  • Investigate whether OSTF1 interaction partners are altered in disease states

• Model system selection:

  • Cell lines expressing endogenous OSTF1 (HeLa, HepG2, MCF7)

  • Primary cells relevant to the disease (e.g., osteoclasts for bone disorders)

  • Animal models with OSTF1 manipulation

  • Patient-derived samples for clinically relevant observations

• Therapeutic potential assessment:

  • Evaluate OSTF1 as a drug target or biomarker

  • Develop approaches to modulate OSTF1 activity in disease-relevant contexts

  • Consider how existing therapies might affect OSTF1 expression or function