FERMT3 Antibody

What is FERMT3 and what cellular functions does it regulate?

FERMT3 (Fermitin family homolog 3, also known as Kindlin-3) is a 72-78 kDa cytoplasmic protein predominantly expressed in hematopoietic cells including B cells, T cells, platelets, neutrophils, and vascular endothelial cells . Structurally, FERMT3 contains:

• A poly-Lys region (aa 147-155)

• A FERM domain (aa 229-558)

• A membrane-anchoring Pleckstrin homology domain (aa 354-457)

FERMT3 functions primarily as an integrin activator, binding to the cytoplasmic tails of β1, β2, and β3 integrins to induce conformational changes that promote cell adhesion . This activation is critical for:

• Integrin-mediated platelet adhesion

• Leukocyte adhesion to endothelial cells

• Leukocyte transmigration

• Proper immune cell function

Deficiencies in FERMT3 result in Leukocyte Adhesion Deficiency type III (LAD-III), a rare autosomal recessive disorder characterized by immune dysfunction and bleeding tendencies .

What applications are FERMT3 antibodies validated for?

FERMT3 antibodies have been validated across multiple applications with specific optimization parameters:

When designing experiments, researchers should note that antibody performance may vary based on:

• Sample preparation method

• Target expression levels

• Antibody lot variability

• Specific tissue/cell type being examined

What is the optimal protocol for Western blotting with FERMT3 antibodies?

For optimal Western blot detection of FERMT3, follow this validated protocol:

• Sample preparation:

  • Use reducing conditions with RIPA or similar lysis buffer

  • Include protease inhibitors to prevent degradation

  • Positive control samples: human platelets, Jurkat cells, THP-1 cells

• Gel electrophoresis and transfer:

  • Load 20-50 μg of total protein per lane

  • Use PVDF membrane for optimal protein binding

• Antibody incubation:

  • Primary antibody: Dilute FERMT3 antibody 1:200-1:1000 in blocking buffer

  • Use 0.5 μg/mL concentration for consistent results

  • Incubate overnight at 4°C for maximum sensitivity

• Detection:

  • Use HRP-conjugated secondary antibody (e.g., Anti-Rabbit IgG)

  • Expected band size: 72-78 kDa

  • For human platelets, a specific band at approximately 80 kDa is typically observed

Troubleshooting note: If multiple bands appear, optimize antibody concentration and incubation time, or consider using more specific FERMT3 antibody clones.

How should FERMT3 antibodies be stored and handled for maximum stability?

Proper storage and handling of FERMT3 antibodies is critical for maintaining reactivity and specificity:

Short-term storage (up to 2 weeks):

• Maintain refrigerated at 2-8°C

• Avoid repeated freeze-thaw cycles

Long-term storage:

• Store at -20°C to -70°C

• For lyophilized antibodies, reconstitute immediately before use

• After reconstitution, antibodies remain stable for approximately:

  • 1 month at 2-8°C under sterile conditions

  • 6 months at -20°C to -70°C under sterile conditions

Buffer conditions:

• Most FERMT3 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

• For diluted working solutions, aliquot and store at recommended temperatures

• Some formulations include preservatives (0.03% Proclin 300) to maintain stability

Experimental note: Performance decreases with each freeze-thaw cycle. For optimal results, prepare small aliquots upon first thaw.

How can researchers validate FERMT3 antibody specificity across different experimental models?

Validating FERMT3 antibody specificity requires multiple complementary approaches:

• Positive and negative control selection:

  • Positive controls: Tissues/cells with confirmed high FERMT3 expression:

    • Human platelets

    • Jurkat cells

    • THP-1 cells

    • J774A.1 cells

    • Raji cells

    • K-562 cells

  • Negative controls:

    • FERMT3 knockout cells

    • Tissues with minimal FERMT3 expression

    • Isotype control antibodies

• Cross-validation methodologies:

  • Compare results from multiple antibody clones targeting different FERMT3 epitopes

  • Perform epitope blocking experiments using recombinant FERMT3 proteins

  • Validate with siRNA or CRISPR knockdown of FERMT3

• Species reactivity verification:

  • Most FERMT3 antibodies show cross-reactivity with human, mouse, and rat samples

  • When testing in new species, validate using sequence homology analysis

  • Human FERMT3 shares 99% amino acid identity with mouse FERMT3 over aa 498-667

• Genetic validation approach:

  • Use samples from patients with known FERMT3 mutations (LAD-III patients)

  • Confirm altered band patterns corresponding to truncated proteins

  • For example, the nonsense variant FERMT3:p.Trp600* results in loss of 68 residues from the C-terminal

What methodological approaches reveal FERMT3's role in integrin activation cascades?

Investigating FERMT3's role in integrin activation requires sophisticated methodological approaches:

• Integrin activation assays:

  • Flow cytometry: Measure activated integrin conformations using conformation-specific antibodies (anti-ITGB1, ITGB2, ITGB3)

  • Adhesion assays: Quantify cell adhesion to integrin substrates (fibronectin, ICAM-1) in FERMT3-manipulated cells

  • FRET-based proximity analysis: Measure FERMT3-integrin interactions in live cells

• FERMT3 structure-function analysis:

  • Generate domain-specific deletions in FERMT3 (especially within F3 subdomain)

  • The F3 subdomain is critical for integrin binding; truncations at Trp600 result in loss of integrin activation capacity

  • Mutations affecting the F3 subdomain disrupt FERMT3's ability to form the integrin-binding pocket

• Phosphorylation site analysis:

  • Investigate the functional significance of phosphorylation at Ser8, Tyr11, and Tyr504

  • Use phospho-specific antibodies to track activation-dependent modifications

• Splice variant characterization:

  • Analyze the 56 kDa splice variant (alternative start site at Met181)

  • Examine the functional consequences of deletion at aa 360-363

Research note: When studying FERMT3 splice variants, Western blotting may reveal additional bands below the main 72-78 kDa band. These represent biologically significant isoforms rather than degradation products.

How does FERMT3 expression pattern change in atherosclerotic plaques and what methodologies best detect these changes?

FERMT3 shows distinct expression patterns in atherosclerotic plaques that can be analyzed through multiple methodological approaches:

• Quantitative expression analysis:

  • qRT-PCR reveals FERMT3 transcript upregulation in arterial plaques (3.99-fold increase, p<0.0001)

  • Both FERMT3 transcript variants are upregulated, with the latter variant specifically associated with unstable plaques (p=0.0004)

• Cell-type specific expression:

  • Confocal immunofluorescence analysis methodology:

    • Double-staining with FERMT3 and cell-type markers

    • CD68 co-localization identifies monocytic origin cells

    • HHF35 co-localization identifies smooth muscle cells

    • Results show FERMT3 predominantly in CD68+ cells, with low co-localization with HHF35

• Correlation with macrophage polarization markers:

  • FERMT3 expression correlates strongly with M2 macrophage markers

  • Hierarchical cluster analysis places FERMT3 in inflammatory/macrophage marker clusters

  • In contrast, FERMT2 clusters with smooth muscle cell markers

• Vascular bed differential expression:

  • Different arterial beds may show varying FERMT3 expression patterns

  • Multi-site sampling is recommended for comprehensive expression profiling

What methodological approaches can identify FERMT3's role in cigarette smoke-induced epithelial-mesenchymal transition?

FERMT3 plays a significant role in cigarette smoke-induced epithelial-mesenchymal transition (EMT) that can be investigated through these methodological approaches:

• Expression analysis in COPD models:

  • RT-PCR and Western blot demonstrate CSE exposure reduces FERMT3 expression in dose- and time-dependent manner in:

    • A549 cells (human type II alveolar cells)

    • HBE cells (human bronchial epithelioid cells)

• Functional manipulation studies:

  • Knockdown methodology: Si-FERMT3 transfection

    • Results in reduced E-cadherin expression

    • Increases Vimentin and Snail expression

  • Overexpression methodology: FERMT3 vector transfection

    • Increases E-cadherin expression

    • Reduces Vimentin and Snail expression

    • Reverses CSE-induced EMT phenotype

• Morphological analysis:

  • Track epithelial-to-mesenchymal morphological transitions

  • FERMT3 overexpression preserves epithelial-like morphology despite CSE exposure

• Migration assays:

  • Wound-healing assay: Quantify scratch closure rates

  • Transwell migration assay: Measure cell migration through 8.0 μm aperture chambers

  • Both assays demonstrate FERMT3's regulatory role in cell migration

• In vivo validation:

  • Immunohistochemical staining of lung tissue

  • Compare FERMT3 expression between control and COPD samples

  • Quantification using Image-Pro 6.1 software

What are the optimal methodological approaches for investigating FERMT3 mutations in Leukocyte Adhesion Deficiency type III?

Investigating FERMT3 mutations in LAD-III requires comprehensive methodological approaches:

• Genetic screening methodology:

  • Whole exome sequencing: Identifies novel variants

    • Example: c.1683-22_1683-19del homozygous variant

    • Non-sense variant: NM_178443.2(FERMT3):c.1800G>A, p.Trp600*

    • Non-stop variant: NM_178443.2(FERMT3):c.2001del p.668Glufs106

  • Segregation analysis: Confirm inheritance patterns in family members

  • Computational prediction: Use tools like CADD, LRT, and MutationTaster to predict variant pathogenicity

• Functional characterization:

  • Flow cytometry: Assess expression of integrin receptors

    • CD11a (integrin αL), CD11b (integrin αM), CD11c (integrin αX)

    • CD18 (integrin β2), CD41 (integrin αIIb), CD61 (integrin β3)

    • Results typically show normal receptor expression but defective activation

  • Platelet function assays: Evaluate aggregation responses to various agonists

  • Leukocyte adhesion assays: Quantify adhesion to endothelial cells or integrin substrates

• Clinical-laboratory correlation:

  • Track specific mutations with clinical phenotypes:

    • Recurrent severe infections

    • Bleeding tendencies

    • Leukocytosis (particularly monocytosis and eosinophilia)

    • Normal inflammatory biomarkers (CRP and ferritin)

• Structural analysis:

  • Model the impact of mutations on protein structure

  • The p.Trp600* truncation results in loss of two α-helices and a β-sheet comprising three β-strands

  • This affects the F3 subdomain that forms the integrin-binding pocket

What considerations are critical when designing immunohistochemistry protocols for FERMT3 detection in different tissue types?

Optimizing immunohistochemistry protocols for FERMT3 detection requires careful consideration of tissue-specific factors:

• Tissue-specific antigen retrieval optimization:

  • For lymphoid tissues: TE buffer pH 9.0 provides optimal epitope exposure

  • Alternative method: Citrate buffer pH 6.0 with extended incubation

  • Formalin-fixed, paraffin-embedded tissues require more rigorous antigen retrieval than frozen sections

• Primary antibody selection and validation:

  • Recommended dilution range: 1:20-1:200 (tissue-dependent)

  • Incubation conditions: 2 hours at room temperature or overnight at 4°C

  • Validation controls:

    • Positive control: Human lymphoma tissue

    • Negative control: Primary antibody omission and isotype control

• Detection system optimization:

  • For brightfield IHC:

    • HRP-conjugated secondary antibody system

    • DAB development time: 3-5 minutes (tissue-dependent)

    • Counterstain with hematoxylin for nuclear definition

  • For immunofluorescence:

    • Fluorophore-conjugated secondary antibodies

    • Include DAPI for nuclear visualization

    • Perform spectral unmixing if tissue has high autofluorescence

• Multiplex immunostaining strategies:

  • For cell-type identification: Co-stain with lineage markers

    • CD68 for monocytic cells

    • HHF35 for smooth muscle cells

    • CD3 for T cells

  • Sequential staining protocol for multiple antibodies:

    • Strip and reprobe or use antibodies from different species

    • Apply antibodies in order of increasing abundance

• Quantification methods:

  • Digital image analysis using Image-Pro 6.1 or similar software

  • Define scoring parameters: intensity, percentage positive cells, subcellular localization

  • For serial sections: register images for comparative analysis

FERMT3 Antibody: Frequently Asked Questions for Researchers

This comprehensive FAQ resource addresses common research questions about FERMT3 antibodies, organized from foundational concepts to advanced research applications. Each section provides methodological guidance based on current scientific knowledge and experimental evidence.

What is FERMT3 and what cellular functions does it regulate?

FERMT3 (Fermitin family homolog 3, also known as Kindlin-3) is a 72-78 kDa cytoplasmic protein predominantly expressed in hematopoietic cells including B cells, T cells, platelets, neutrophils, and vascular endothelial cells . Structurally, FERMT3 contains:

• A poly-Lys region (aa 147-155)

• A FERM domain (aa 229-558)

• A membrane-anchoring Pleckstrin homology domain (aa 354-457)

FERMT3 functions primarily as an integrin activator, binding to the cytoplasmic tails of β1, β2, and β3 integrins to induce conformational changes that promote cell adhesion . This activation is critical for:

• Integrin-mediated platelet adhesion

• Leukocyte adhesion to endothelial cells

• Leukocyte transmigration

• Proper immune cell function

Deficiencies in FERMT3 result in Leukocyte Adhesion Deficiency type III (LAD-III), a rare autosomal recessive disorder characterized by immune dysfunction and bleeding tendencies .

What applications are FERMT3 antibodies validated for?

FERMT3 antibodies have been validated across multiple applications with specific optimization parameters:

When designing experiments, researchers should note that antibody performance may vary based on:

• Sample preparation method

• Target expression levels

• Antibody lot variability

• Specific tissue/cell type being examined

What is the optimal protocol for Western blotting with FERMT3 antibodies?

For optimal Western blot detection of FERMT3, follow this validated protocol:

• Sample preparation:

  • Use reducing conditions with RIPA or similar lysis buffer

  • Include protease inhibitors to prevent degradation

  • Positive control samples: human platelets, Jurkat cells, THP-1 cells

• Gel electrophoresis and transfer:

  • Load 20-50 μg of total protein per lane

  • Use PVDF membrane for optimal protein binding

• Antibody incubation:

  • Primary antibody: Dilute FERMT3 antibody 1:200-1:1000 in blocking buffer

  • Use 0.5 μg/mL concentration for consistent results

  • Incubate overnight at 4°C for maximum sensitivity

• Detection:

  • Use HRP-conjugated secondary antibody (e.g., Anti-Rabbit IgG)

  • Expected band size: 72-78 kDa

  • For human platelets, a specific band at approximately 80 kDa is typically observed

Troubleshooting note: If multiple bands appear, optimize antibody concentration and incubation time, or consider using more specific FERMT3 antibody clones.

How should FERMT3 antibodies be stored and handled for maximum stability?

Proper storage and handling of FERMT3 antibodies is critical for maintaining reactivity and specificity:

Short-term storage (up to 2 weeks):

• Maintain refrigerated at 2-8°C

• Avoid repeated freeze-thaw cycles

Long-term storage:

• Store at -20°C to -70°C

• For lyophilized antibodies, reconstitute immediately before use

• After reconstitution, antibodies remain stable for approximately:

  • 1 month at 2-8°C under sterile conditions

  • 6 months at -20°C to -70°C under sterile conditions

Buffer conditions:

• Most FERMT3 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

• For diluted working solutions, aliquot and store at recommended temperatures

• Some formulations include preservatives (0.03% Proclin 300) to maintain stability

Experimental note: Performance decreases with each freeze-thaw cycle. For optimal results, prepare small aliquots upon first thaw.

How can researchers validate FERMT3 antibody specificity across different experimental models?

Validating FERMT3 antibody specificity requires multiple complementary approaches:

• Positive and negative control selection:

  • Positive controls: Tissues/cells with confirmed high FERMT3 expression:

    • Human platelets

    • Jurkat cells

    • THP-1 cells

    • J774A.1 cells

    • Raji cells

    • K-562 cells

  • Negative controls:

    • FERMT3 knockout cells

    • Tissues with minimal FERMT3 expression

    • Isotype control antibodies

• Cross-validation methodologies:

  • Compare results from multiple antibody clones targeting different FERMT3 epitopes

  • Perform epitope blocking experiments using recombinant FERMT3 proteins

  • Validate with siRNA or CRISPR knockdown of FERMT3

• Species reactivity verification:

  • Most FERMT3 antibodies show cross-reactivity with human, mouse, and rat samples

  • When testing in new species, validate using sequence homology analysis

  • Human FERMT3 shares 99% amino acid identity with mouse FERMT3 over aa 498-667

• Genetic validation approach:

  • Use samples from patients with known FERMT3 mutations (LAD-III patients)

  • Confirm altered band patterns corresponding to truncated proteins

  • For example, the nonsense variant FERMT3:p.Trp600* results in loss of 68 residues from the C-terminal

What methodological approaches reveal FERMT3's role in integrin activation cascades?

Investigating FERMT3's role in integrin activation requires sophisticated methodological approaches:

• Integrin activation assays:

  • Flow cytometry: Measure activated integrin conformations using conformation-specific antibodies (anti-ITGB1, ITGB2, ITGB3)

  • Adhesion assays: Quantify cell adhesion to integrin substrates (fibronectin, ICAM-1) in FERMT3-manipulated cells

  • FRET-based proximity analysis: Measure FERMT3-integrin interactions in live cells

• FERMT3 structure-function analysis:

  • Generate domain-specific deletions in FERMT3 (especially within F3 subdomain)

  • The F3 subdomain is critical for integrin binding; truncations at Trp600 result in loss of integrin activation capacity

  • Mutations affecting the F3 subdomain disrupt FERMT3's ability to form the integrin-binding pocket

• Phosphorylation site analysis:

  • Investigate the functional significance of phosphorylation at Ser8, Tyr11, and Tyr504

  • Use phospho-specific antibodies to track activation-dependent modifications

• Splice variant characterization:

  • Analyze the 56 kDa splice variant (alternative start site at Met181)

  • Examine the functional consequences of deletion at aa 360-363

Research note: When studying FERMT3 splice variants, Western blotting may reveal additional bands below the main 72-78 kDa band. These represent biologically significant isoforms rather than degradation products.

How does FERMT3 expression pattern change in atherosclerotic plaques and what methodologies best detect these changes?

FERMT3 shows distinct expression patterns in atherosclerotic plaques that can be analyzed through multiple methodological approaches:

• Quantitative expression analysis:

  • qRT-PCR reveals FERMT3 transcript upregulation in arterial plaques (3.99-fold increase, p<0.0001)

  • Both FERMT3 transcript variants are upregulated, with the latter variant specifically associated with unstable plaques (p=0.0004)

• Cell-type specific expression:

  • Confocal immunofluorescence analysis methodology:

    • Double-staining with FERMT3 and cell-type markers

    • CD68 co-localization identifies monocytic origin cells

    • HHF35 co-localization identifies smooth muscle cells

    • Results show FERMT3 predominantly in CD68+ cells, with low co-localization with HHF35

• Correlation with macrophage polarization markers:

  • FERMT3 expression correlates strongly with M2 macrophage markers

  • Hierarchical cluster analysis places FERMT3 in inflammatory/macrophage marker clusters

  • In contrast, FERMT2 clusters with smooth muscle cell markers

• Vascular bed differential expression:

  • Different arterial beds may show varying FERMT3 expression patterns

  • Multi-site sampling is recommended for comprehensive expression profiling

What methodological approaches can identify FERMT3's role in cigarette smoke-induced epithelial-mesenchymal transition?

FERMT3 plays a significant role in cigarette smoke-induced epithelial-mesenchymal transition (EMT) that can be investigated through these methodological approaches:

• Expression analysis in COPD models:

  • RT-PCR and Western blot demonstrate CSE exposure reduces FERMT3 expression in dose- and time-dependent manner in:

    • A549 cells (human type II alveolar cells)

    • HBE cells (human bronchial epithelioid cells)

• Functional manipulation studies:

  • Knockdown methodology: Si-FERMT3 transfection

    • Results in reduced E-cadherin expression

    • Increases Vimentin and Snail expression

  • Overexpression methodology: FERMT3 vector transfection

    • Increases E-cadherin expression

    • Reduces Vimentin and Snail expression

    • Reverses CSE-induced EMT phenotype

• Morphological analysis:

  • Track epithelial-to-mesenchymal morphological transitions

  • FERMT3 overexpression preserves epithelial-like morphology despite CSE exposure

• Migration assays:

  • Wound-healing assay: Quantify scratch closure rates

  • Transwell migration assay: Measure cell migration through 8.0 μm aperture chambers

  • Both assays demonstrate FERMT3's regulatory role in cell migration

• In vivo validation:

  • Immunohistochemical staining of lung tissue

  • Compare FERMT3 expression between control and COPD samples

  • Quantification using Image-Pro 6.1 software

What are the optimal methodological approaches for investigating FERMT3 mutations in Leukocyte Adhesion Deficiency type III?

Investigating FERMT3 mutations in LAD-III requires comprehensive methodological approaches:

• Genetic screening methodology:

  • Whole exome sequencing: Identifies novel variants

    • Example: c.1683-22_1683-19del homozygous variant

    • Non-sense variant: NM_178443.2(FERMT3):c.1800G>A, p.Trp600*

    • Non-stop variant: NM_178443.2(FERMT3):c.2001del p.668Glufs106

  • Segregation analysis: Confirm inheritance patterns in family members

  • Computational prediction: Use tools like CADD, LRT, and MutationTaster to predict variant pathogenicity

• Functional characterization:

  • Flow cytometry: Assess expression of integrin receptors

    • CD11a (integrin αL), CD11b (integrin αM), CD11c (integrin αX)

    • CD18 (integrin β2), CD41 (integrin αIIb), CD61 (integrin β3)

    • Results typically show normal receptor expression but defective activation

  • Platelet function assays: Evaluate aggregation responses to various agonists

  • Leukocyte adhesion assays: Quantify adhesion to endothelial cells or integrin substrates

• Clinical-laboratory correlation:

  • Track specific mutations with clinical phenotypes:

    • Recurrent severe infections

    • Bleeding tendencies

    • Leukocytosis (particularly monocytosis and eosinophilia)

    • Normal inflammatory biomarkers (CRP and ferritin)

• Structural analysis:

  • Model the impact of mutations on protein structure

  • The p.Trp600* truncation results in loss of two α-helices and a β-sheet comprising three β-strands

  • This affects the F3 subdomain that forms the integrin-binding pocket

What considerations are critical when designing immunohistochemistry protocols for FERMT3 detection in different tissue types?

Optimizing immunohistochemistry protocols for FERMT3 detection requires careful consideration of tissue-specific factors:

• Tissue-specific antigen retrieval optimization:

  • For lymphoid tissues: TE buffer pH 9.0 provides optimal epitope exposure

  • Alternative method: Citrate buffer pH 6.0 with extended incubation

  • Formalin-fixed, paraffin-embedded tissues require more rigorous antigen retrieval than frozen sections

• Primary antibody selection and validation:

  • Recommended dilution range: 1:20-1:200 (tissue-dependent)

  • Incubation conditions: 2 hours at room temperature or overnight at 4°C

  • Validation controls:

    • Positive control: Human lymphoma tissue

    • Negative control: Primary antibody omission and isotype control

• Detection system optimization:

  • For brightfield IHC:

    • HRP-conjugated secondary antibody system

    • DAB development time: 3-5 minutes (tissue-dependent)

    • Counterstain with hematoxylin for nuclear definition

  • For immunofluorescence:

    • Fluorophore-conjugated secondary antibodies

    • Include DAPI for nuclear visualization

    • Perform spectral unmixing if tissue has high autofluorescence

• Multiplex immunostaining strategies:

  • For cell-type identification: Co-stain with lineage markers

    • CD68 for monocytic cells

    • HHF35 for smooth muscle cells

    • CD3 for T cells

  • Sequential staining protocol for multiple antibodies:

    • Strip and reprobe or use antibodies from different species

    • Apply antibodies in order of increasing abundance

• Quantification methods:

  • Digital image analysis using Image-Pro 6.1 or similar software

  • Define scoring parameters: intensity, percentage positive cells, subcellular localization

  • For serial sections: register images for comparative analysis