FLOT2 Antibody

What is FLOT2 and why is it significant in cellular biology?

FLOT2 (flotillin 2, also named reggie-2) is a key component of lipid rafts located in the plasma membrane and is implicated in modulating various cellular processes . With a protein length of 428 amino acids and a molecular mass of approximately 47.1 kDa, FLOT2 is also known by several synonyms including ECS-1, ESA, and epidermal surface antigen 1 . FLOT2 plays critical roles in membrane organization, receptor trafficking, and signal transduction pathways relevant to both normal physiology and disease states.

What are the validated applications for FLOT2 antibodies in research?

FLOT2 antibodies have been successfully applied in multiple experimental techniques including:

• Immunohistochemistry (IHC) for tissue expression analysis

• Western blotting for protein expression quantification

• Immunoprecipitation for protein-protein interaction studies

• Flow cytometry for cell-based assays

Each application requires specific antibody validation to ensure reliability and reproducibility of results across different experimental conditions .

How is FLOT2 expression typically analyzed in tissue samples?

FLOT2 expression in tissue samples is commonly assessed through immunohistochemical staining followed by semi-quantitative scoring. The standard procedure involves:

• Sample fixation in 4% formaldehyde solution

• Paraffin embedding and sectioning (4-μm thickness)

• Antigen retrieval using citrate buffer (pH 6.0)

• Blocking with bovine serum albumin

• Overnight incubation with anti-FLOT2 antibody

• Signal development using horseradish peroxidase-conjugated secondary antibody

Expression is typically scored based on both staining intensity (0-3) and proportion of positive cells (1-4), with the product of these scores determining high versus low expression status .

What is the optimal protocol for detecting FLOT2 and its phosphorylation status by Western blot?

For optimal Western blot detection of FLOT2, especially when studying associated signaling events:

• Cell preparation:

  • Rest cells in plain media at 37°C for 2 hours prior to stimulation

  • Stimulate cells as needed for your experimental design

• Lysis methods (multiple options):

  • Direct addition of 10× RIPA buffer followed by 30-minute lysis on ice

  • Immediate addition of 1× RIPA buffer after media removal

  • Direct addition of 4× LDS with 20× DTT (1M)

• Sample processing:

  • For RIPA-lysed samples: centrifuge at 18,800 g for 15 minutes at 4°C

  • For direct LDS lysis: boil for 10 minutes at 70-95°C

• Loading normalization:

  • Use equal cell numbers or protein amounts (validated by BCA assay)

  • Normalize to appropriate housekeeping proteins

This protocol has been validated for studying FLOT2's relationship with T cell receptor signaling pathways .

How can researchers effectively study FLOT2 interactions with other proteins?

Studying FLOT2 protein interactions requires careful experimental design:

• Immunoprecipitation approach:

  • Prepare cell lysates under non-denaturing conditions

  • Immunoprecipitate FLOT2 using validated antibodies

  • Analyze co-precipitated proteins by Western blot

• Stimulus-dependent interactions:

  • Compare resting versus stimulated conditions (e.g., with EGF)

  • Track temporal dynamics of interactions

• Controls to include:

  • FLOT2 knockout cells as negative controls

  • Isotype control antibodies

  • Input samples to verify protein expression levels

Using this approach, researchers have identified important interactions between FLOT2 and proteins like Cbl, which decrease upon EGF stimulation .

What considerations are important when designing FLOT2 knockout or knockdown experiments?

When manipulating FLOT2 expression for functional studies:

• Validation requirements:

  • Confirm complete protein depletion by Western blot

  • Create proper control lines (e.g., targeting non-expressed genes like eGFP)

  • Verify specificity of phenotypes through rescue experiments

• Experimental design considerations:

  • Consider cell type-specific effects (expression varies across tissues)

  • Account for potential compensatory mechanisms (especially by FLOT1)

  • Analyze multiple clones to rule out off-target effects

• Functional readouts:

  • Assess receptor activation (phosphorylation status)

  • Evaluate downstream signaling pathway activation

  • Monitor receptor trafficking and degradation rates

These approaches have revealed that FLOT2 negatively regulates EGFR activation and dimerization, affecting subsequent ubiquitination, endosomal trafficking, and degradation .

How does FLOT2 expression correlate with cancer progression and prognosis?

Multiple studies have established strong correlations between FLOT2 expression and cancer outcomes:

FLOT2 serves as an independent prognostic predictor in multivariate analyses, particularly significant in advanced cancer stages (pT3-4 and AJCC stage III-IV) .

What molecular mechanisms underlie FLOT2's contribution to cancer progression?

FLOT2 promotes cancer progression through multiple mechanisms:

• TGF-β pathway regulation:

  • FLOT2 positively regulates CD109 expression

  • CD109 negatively regulates TGF-β signaling

  • This leads to inactivation of the TGF-β signaling pathway in nasopharyngeal carcinoma

• EGFR signaling modulation:

  • FLOT2 knockdown increases basal EGFR phosphorylation

  • This results in constitutive activation of downstream MAPK signaling

  • Increased EGFR ubiquitination and association with Cbl occurs upon FLOT2 depletion

These mechanisms highlight FLOT2's complex roles in regulating critical cancer-associated signaling pathways .

How should researchers score and interpret FLOT2 immunohistochemical staining in cancer tissues?

For standardized FLOT2 expression analysis in cancer:

• Scoring methodology:

  • Proportion score: 1 (<10% positive cells), 2 (10-50%), 3 (50-75%), 4 (>75%)

  • Intensity score: 0 (no staining), 1 (weak/light yellow), 2 (moderate/yellow-brown), 3 (strong/brown)

  • Staining index = proportion score × intensity score

• Threshold determination:

  • Optimal cutoffs should be identified based on survival analysis

  • Typically, staining index ≥6 defines high expression

  • Staining index ≤4 indicates low expression

• Statistical analysis:

  • Compare expression between tumor and adjacent normal tissues using paired t-tests

  • Correlate with clinicopathological parameters using Chi-square tests

  • Perform survival analysis using Kaplan-Meier method and log-rank tests

  • Conduct multivariate analysis using Cox proportional hazards regression

This approach enables robust evaluation of FLOT2 as a prognostic biomarker in cancer research .

How does FLOT2 influence T cell function and antigen sensitivity?

Recent research has revealed FLOT2's significant role in T cell biology:

• T cell antigen sensitivity:

  • Deleting FLOT2 increases T cell sensitivity to antigen

  • This results in enhanced TCR signaling and functionality

  • FLOT2-deficient T cells show increased activation even with weak stimulation

• Transcriptional impacts:

  • Single-cell RNA sequencing reveals distinct activation patterns

  • FLOT2-deficient CD4+ T cells show higher occupancy in the activated cluster following weak stimulation

  • FLOT2 deletion alters distribution across activation clusters identified by marker genes

• Functional implications:

  • FLOT2 appears to regulate surface TCR clustering

  • Targeting FLOT2 may enhance T cell reactivity in diseases with weak antigenicity

  • This approach holds promise for cancer immunotherapy and chronic infection treatment

These findings suggest important therapeutic applications for modulating FLOT2 in T cell-based treatments .

What experimental approaches are recommended for analyzing FLOT2's role in T cell signaling?

To comprehensively analyze FLOT2's impact on T cell signaling:

• Genetic models:

  • Generate conditional knockouts (e.g., Flot2CD4 for T cell-specific deletion)

  • Compare knockout cells with wild-type controls under identical conditions

• Activation analysis:

  • Use varying concentrations of αCD3 antibody (0, 0.25, 1.0 μg/mL) to create different stimulation strengths

  • Measure early activation markers (CD69, CD25) by flow cytometry

  • Assess phosphorylation of signaling molecules after brief (3-minute) stimulation

• Transcriptomic approaches:

  • Perform single-cell RNA sequencing to identify activation states

  • Use clustering algorithms (e.g., Leiden) to identify functional states

  • Analyze distribution across activation clusters as a measure of sensitivity

This multilevel approach provides robust insights into FLOT2's regulatory functions in T cell biology .

How can researchers validate the specificity of FLOT2 antibodies?

Ensuring antibody specificity is crucial for reliable FLOT2 research:

• Genetic validation:

  • Test antibodies in FLOT2 knockout or knockdown models

  • Complete protein loss should result in absence of signal

• Multiple detection methods:

  • Compare results across techniques (Western blot, IHC, flow cytometry)

  • Verify consistent expression patterns across methods

• Critical controls:

  • Include isotype control antibodies

  • Use blocking peptides to confirm epitope specificity

  • Compare multiple antibody clones targeting different epitopes

• Species cross-reactivity:

  • Verify species-specific reactivity when working with models from different organisms

  • Consider sequence homology when interpreting results across species

These validation steps ensure accurate attribution of experimental results to FLOT2-specific effects .

What are common pitfalls in FLOT2 detection and how can they be addressed?

Researchers should be aware of these common challenges:

• Membrane protein preservation:

  • Ensure proper sample preparation to maintain membrane integrity

  • Consider specialized lysis buffers for lipid raft proteins

• Cross-reactivity concerns:

  • FLOT1 and FLOT2 share structural similarities

  • Verify antibody specificity against both proteins

• Expression variability:

  • FLOT2 expression varies across tissues and cell types

  • Include appropriate positive and negative control samples

• Post-translational modifications:

  • Consider how modifications might affect antibody recognition

  • Use phospho-specific antibodies when studying signaling events

Addressing these considerations enhances experimental reliability and reproducibility in FLOT2 research .