TFE3 Antibody

What is TFE3 and why is it significant in cellular biology?

TFE3 (Transcription Factor Binding To IGHM Enhancer 3) is a basic helix-loop-helix domain-containing transcription factor that belongs to the MiT/TFE family. It functions as a master regulator of lysosomal biogenesis and immune response. TFE3 specifically recognizes and binds E-box sequences (5'-CANNTG-3') and the CLEAR-box sequence (5'-GTCACGTGAC-3') present in the regulatory regions of many lysosomal genes .

TFE3 plays crucial roles in:

• Lysosomal gene expression and biogenesis

• Cellular response to nutrient availability

• Maintaining pluripotency in embryonic and hematopoietic stem cells

• Regulation of browning in adipose tissue

• T-cell-dependent antibody responses

Its activity is primarily regulated through mTOR-dependent phosphorylation, which determines its subcellular localization and function .

What are the typical reactivity profiles of commercial TFE3 antibodies?

Most commercially available TFE3 antibodies demonstrate:

Most polyclonal antibodies are generated in rabbits using synthetic peptides corresponding to specific amino acid regions of human TFE3. For example, Abcepta's polyclonal antibody (AP18317b) targets the C-terminal region (amino acids 489-516) , while Abcam's antibody (ab245454) targets amino acids 450-500 .

How do TFE3 antibodies perform in diagnostic applications for TFE3-associated neoplasms?

TFE3 antibodies demonstrate variable performance in different tumor types:

This table demonstrates the high sensitivity and specificity of TFE3 immunostaining for certain tumor types, particularly alveolar soft part sarcoma and pediatric renal cell carcinoma with TFE3 gene fusions .

How can researchers differentiate between wild-type TFE3 and TFE3 fusion proteins using antibodies?

Distinguishing between wild-type TFE3 and fusion proteins requires careful methodological considerations:

• Antibody selection: Use antibodies targeting the C-terminal region of TFE3, which is typically retained in fusion proteins .

• Staining pattern analysis:

  • Wild-type TFE3: Primarily cytoplasmic in nutrient-rich conditions, with nuclear translocation during starvation

  • Fusion proteins: Often show constitutive nuclear localization due to loss of regulatory domains

• Controls: Include known positive controls (such as alveolar soft part sarcoma) and negative controls .

• Complementary techniques: Confirm antibody findings with FISH or molecular testing, as TFE3 immunostaining alone lacks sufficient specificity for definitive diagnosis of translocation status .

Research shows that TFE3-splicing factor fusions drive transformation of kidney cells and promote distinct oncogenic phenotypes in a fusion partner-dependent manner. These fusion proteins differentially alter the transcriptome and RNA splicing landscape, activating different oncogenic pathways .

What factors affect subcellular localization of TFE3 and how does this impact antibody-based detection?

TFE3 subcellular localization is dynamically regulated by multiple factors:

• Nutrient availability: In nutrient-rich conditions, TFE3 is predominantly cytoplasmic; during starvation, it translocates to the nucleus .

• mTOR pathway: When nutrients are present, TFE3 is recruited to the lysosomal membrane via association with GDP-bound RagC/RRAGC (or RagD/RRAGD) and phosphorylated by mTOR .

• Phosphorylation status: Phosphorylation by mTOR prevents nuclear translocation and promotes ubiquitination and degradation. Dephosphorylation during starvation or lysosomal stress enables nuclear translocation .

• 14-3-3 interaction: Phosphorylated TFE3 (particularly at Ser321) binds to 14-3-3 proteins, which sequester it in the cytosol. This interaction is abolished by mTORC1 inactivation .

For accurate antibody-based detection:

• Fixation methods must preserve both nuclear and cytoplasmic pools of TFE3

• Researchers should control for nutrient conditions that may affect TFE3 localization

• Consider dual staining with phospho-specific antibodies to distinguish active from inactive forms

• Document fixation time and conditions, as these significantly impact subcellular preservation

What are the optimal methodological approaches for detecting TFE3 in immunohistochemical applications?

For optimal TFE3 detection in IHC applications:

• Tissue preparation:

  • Formalin-fixed, paraffin-embedded tissue sections

  • Heat-induced epitope retrieval is recommended over enzymatic retrieval

• Staining protocol optimization:

  • Antibody concentration: Initial validation using dilution series

  • Incubation time and temperature: Typically overnight at 4°C for optimal signal-to-noise ratio

  • Detection system: Use high-sensitivity detection systems for low-abundance targets

• Controls and validation:

  • Positive control: Alveolar soft part sarcoma shows consistent strong nuclear positivity

  • Negative control: Normal tissues with known absence of TFE3 expression

  • Internal control: Non-neoplastic cells within the specimen should show expected patterns

• Interpretation guidelines:

  • Diffuse strong nuclear expression is considered positive in neoplastic cells

  • Weak or focal staining requires careful interpretation and confirmation with molecular techniques

  • Cytoplasmic staining patterns should be evaluated in the context of mTOR pathway activity

How does TFE3 regulate lysosomal biogenesis and what methods can researchers use to study this function?

TFE3 regulates lysosomal biogenesis through several mechanisms:

• CLEAR element binding: TFE3 binds to Coordinated Lysosomal Expression And Regulation (CLEAR) elements (5'-GTCACGTGAC-3') in promoters of lysosomal genes .

• Target gene activation: TFE3 activates genes involved in lysosomal function, including:

  • Cathepsin D (CTSD)

  • Mucolipin-1 (MCOLN1)

  • V-type proton ATPase components (ATP6V0D2, ATP6V1C1)

  • Lysosomal hydrolases (GBA, GLA, HEXA)

• Increased lysosomal number: Overexpression of TFE3 leads to increased numbers of LAMP1-positive structures (from 383±90 to 722±202 lysosomes per cell in ARPE-19 cells) .

To study these functions, researchers can use:

• CLEARoptimized reporter system: A biosensor consisting of six CLEAR motifs driving luciferase and tdTomato expression to quantify TFE3 activity in cells and animals .

• LAMP1 quantification: Immunofluorescence staining of LAMP1 followed by confocal microscopy and quantitative image analysis .

• Luciferase reporter assays: Using promoters of lysosomal genes (like MCOLN1) with wild-type or mutated CLEAR elements to assess TFE3 transactivation potential .

• Lysosomal enzyme activity assays: Measuring activity of acid phosphatase and other lysosomal hydrolases in cells and culture medium .

What is the relationship between TFE3 and nerve regeneration, and how can antibodies help investigate this function?

Recent research has revealed an important role for TFE3 in nerve regeneration processes:

• Upregulation after injury: TFE3 expression increases significantly following nerve damage, suggesting its involvement in repair mechanisms .

• Schwann cell function: TFE3 (along with TFEB) governs repair Schwann cell generation and function following peripheral nerve injury .

• Regulatory mechanisms: TFE3 may regulate genes associated with axon regeneration and Schwann cell activation, essential components of the nerve repair process .

Anti-TFE3 antibodies have been instrumental in investigating these functions through:

• Protein expression analysis: Detecting upregulation of TFE3 in injured nerves using Western blotting

• Cellular localization: Determining precise location of TFE3 activity in nerve tissues through immunohistochemistry and immunofluorescence

• Chromatin immunoprecipitation: Identifying TFE3 target genes in neural cells during regeneration

• Co-immunoprecipitation: Investigating protein-protein interactions involved in TFE3-mediated nerve repair

These methodologies allow researchers to elucidate the molecular mechanisms by which TFE3 influences nerve injury responses, potentially leading to improved therapeutic approaches for nerve regeneration.

How does the metabolic state of cells impact TFE3 function and antibody detection?

TFE3 functions as a nutrient-responsive transcription factor whose activity is tightly regulated by cellular metabolic state:

• Nutrient sensing:

  • In nutrient-rich conditions: TFE3 is phosphorylated by mTOR, bound to 14-3-3 proteins, and sequestered in the cytoplasm

  • During starvation: mTOR inhibition leads to TFE3 dephosphorylation and nuclear translocation

• Metabolic reprogramming in cancer:

  • TFE3 fusion-driven renal cell carcinomas exhibit a shift toward oxidative phosphorylation (OXPHOS), contrasting with the glycolytic metabolism typical of most renal cancers

  • This metabolic rewiring, combined with elevated glutathione levels, renders these tumors sensitive to reductive stress

• Autophagy regulation:

  • TFE3 activates autophagy-related genes during nutrient deprivation

  • This function is critical for cellular adaptation to metabolic stress

Considerations for antibody-based detection:

• Sample preparation: The metabolic state of cells/tissues at the time of fixation influences TFE3 localization and potentially epitope accessibility

• Fasting/feeding status: For in vivo studies, the nutritional status of animals may significantly impact TFE3 localization

• Tissue-specific metabolism: Different tissues exhibit varying baseline levels of mTOR activity, affecting TFE3 phosphorylation and localization

• Cancer metabolism: TFE3 fusion-positive tumors may show altered localization patterns due to metabolic reprogramming

How can TFE3 antibodies be used to investigate TFE3 fusion-driven oncogenesis?

TFE3 antibodies are valuable tools for investigating the mechanisms of TFE3 fusion-driven oncogenesis:

• Chromatin Immunoprecipitation and Sequencing (ChIP-Seq):

  • Can identify genomic binding sites of TFE3 fusion proteins

  • Research has identified 1,347 TFE3 fusion peaks shared across three tRCC cell lines

  • Enriched pathways in TFE3 fusion-bound regions include lysosomal biogenesis, autophagy, and mTOR signaling

• Fusion partner identification:

  • Different staining patterns may suggest specific fusion partners

  • Various fusion partners have been identified, including ASPL, PRCC, NONO, and SFPQ

  • Each fusion produces distinct oncogenic phenotypes and transcriptomic alterations

• Therapeutic target identification:

  • TFE3 dimerization has been identified as a potential therapeutic target

  • High-throughput screening using FRET technology identified compounds that inhibit TFE3-SF dimerization

  • The antihistamine terfenadine was found to decrease cell proliferation and reduce in vivo tumor growth of tRCC

• Metabolic phenotyping:

  • TFE3 fusion proteins drive a shift toward oxidative phosphorylation in tRCC

  • This metabolic reprogramming creates specific vulnerabilities that can be exploited therapeutically

What are the technical challenges in using TFE3 antibodies for diagnostic purposes, and how can they be overcome?

Several technical challenges exist when using TFE3 antibodies for diagnostic purposes:

• Variable sensitivity and specificity:

  • Sensitivity and specificity depend on antibody type (polyclonal versus monoclonal)

  • Tissue fixation and staining conditions significantly impact results

  • While TFE3 immunostaining is valuable as a screening tool, it lacks the specificity of FISH studies

• False positives and negatives:

  • Some tumors may show weak non-specific nuclear staining

  • Fixation artifacts can lead to false-negative results

  • Cross-reactivity with other MiT family members may occur

• Threshold determination:

  • No standardized criteria exist for what constitutes "positive" staining

  • Variability in interpretation between pathologists

To overcome these challenges:

• Standardized protocols: Implement rigorous, standardized protocols for fixation, antigen retrieval, and staining

• Complementary techniques: Use TFE3 immunostaining as a screening tool, followed by FISH or molecular testing for confirmation

• Positive controls: Include known TFE3 fusion-positive cases (alveolar soft part sarcoma) as positive controls

• Monoclonal antibodies: Consider using newer generation monoclonal antibodies with improved specificity

• Digital pathology: Employ quantitative image analysis to establish objective thresholds for positivity

How can researchers study TFE3 activity in real-time using advanced reporter systems?

The recently developed CLEARoptimized reporter system offers an innovative approach to studying TFE3 activity in real-time:

• Reporter design:

  • Contains six coordinated lysosomal expression and regulation (CLEAR) motifs identified through bioinformatic analysis of 128 TFEB-target gene promoters

  • Drives expression of luciferase and tdTomato reporter genes

  • Enables quantification of TFE3 activity in cells and animals through optical imaging and biochemical assays

• Applications:

  • Cellular studies: Monitor TFE3 activity in response to nutrient availability, drugs, or genetic perturbations

  • In vivo imaging: Track TFE3 activity in transgenic reporter mice using non-invasive bioluminescence imaging

  • Drug screening: Identify compounds that modulate TFE3 activity

  • Disease models: Study TFE3 dysregulation in models of lysosomal storage disorders, neurodegeneration, or cancer

• Advantages:

  • Non-invasive monitoring of TFE3 activity

  • Compatible with high-throughput screening approaches

  • Allows longitudinal studies in the same animal

  • Provides spatial information about TFE3 activity in different tissues

• Methodology:

  • Generate stable cell lines or transgenic mice expressing the CLEARoptimized reporter

  • Apply stimuli known to modulate TFE3 activity (e.g., starvation, mTOR inhibitors)

  • Measure luciferase activity through bioluminescence imaging or biochemical assays

  • Quantify tdTomato expression through fluorescence microscopy or flow cytometry

This reporter system represents a significant advancement in the field, enabling the pharmacological profiling of TFE3-mediated transcription and facilitating the development of targeted therapeutics.

What emerging applications of TFE3 antibodies are being developed for therapeutic targeting?

Recent research has identified several promising therapeutic approaches targeting TFE3:

• TFE3 dimerization inhibitors:

  • High-throughput high-content screening (HTHCS) combined with FRET technology has identified compounds that inhibit TFE3-splicing factor dimerization

  • The antihistamine terfenadine showed efficacy in decreasing cell proliferation and reducing in vivo tumor growth in tRCC models

• Metabolic vulnerability targeting:

  • TFE3 fusion-driven cancers exhibit distinct metabolic profiles, including increased oxidative phosphorylation

  • This creates specific vulnerabilities to reductive stress that can be exploited therapeutically

  • CRISPR screening has identified tRCC-selective vulnerabilities linked to this metabolic state, including EGLN1

• mTOR pathway modulation:

  • Since TFE3 activity is regulated by mTOR-dependent phosphorylation, mTOR inhibitors may affect TFE3 localization and function

  • Targeted approaches to modulate specific TFE3 phosphorylation sites could provide selective therapeutic effects

• Splicing modulation:

  • TFE3-splicing factor fusions possess both transcription and splicing factor functions

  • Targeting the altered splicing patterns in TFE3 fusion-positive tumors represents a novel therapeutic avenue

TFE3 antibodies will be crucial for validating these therapeutic approaches through:

• Target engagement studies

• Pharmacodynamic biomarker development

• Patient stratification for clinical trials

How can researchers optimize TFE3 antibody selection for specific experimental applications?

Selecting the optimal TFE3 antibody requires careful consideration of experimental goals:

• Epitope location:

  • N-terminal antibodies: Detect wild-type TFE3 but may miss some fusion proteins where the N-terminus is replaced

  • C-terminal antibodies: Detect both wild-type and fusion proteins as the C-terminus is usually retained

  • Middle region antibodies: May have varying specificity depending on fusion breakpoints

• Antibody format:

  • Monoclonal antibodies: Higher specificity but may miss some epitope variants

  • Polyclonal antibodies: Higher sensitivity but potential for cross-reactivity

  • Rabbit-derived antibodies have shown good performance in detecting TFE3

• Application-specific considerations:

  Application	Recommended Antibody Characteristics
  IHC	Fixed epitope-tolerant antibodies validated with proper controls
  Western blot	Antibodies recognizing denatured epitopes (typically linear)
  ChIP-seq	High-specificity antibodies with minimal background binding
  Flow cytometry	Antibodies validated for native protein detection

• Validation methods:

  • Use multiple antibodies targeting different epitopes

  • Include genetic knockdown/knockout controls

  • Compare results with orthogonal detection methods

  • Test in known positive and negative cell lines or tissues

• Experimental documentation:

  • Record antibody catalog number, lot, dilution, and incubation conditions

  • Document optimization steps and validation results

  • Consider antibody validation principles from initiatives like the Antibody Validation Database

What novel insights into TFE3 biology have been revealed through recent antibody-based studies?

Recent antibody-based studies have uncovered several novel aspects of TFE3 biology:

• Role in nerve regeneration:

  • TFE3 is significantly upregulated following nerve damage

  • It plays a crucial role in Schwann cell activation and function during nerve repair

  • This represents a novel therapeutic target for enhancing nerve regeneration

• Metabolic rewiring in cancer:

  • TFE3 fusion proteins transcriptionally rewire tRCCs toward oxidative phosphorylation

  • This contrasts with the highly glycolytic metabolism of most renal cancers

  • The combined OXPHOS program and heightened glutathione levels create a specific vulnerability to reductive stress

• Complex regulation by Rag GTPases:

  • TFE3 interacts with Rag heterodimers in their active conformation (RagB GTP/RagD GDP)

  • This interaction is mediated by a conserved Rag-binding motif in TFE3

  • Mutation of this motif dramatically reduces the interaction between TFE3 and active Rags

• Independent function from TFEB:

  • Despite their structural similarity, TFE3 promotes expression of lysosomal genes independently of TFEB

  • TFE3 overexpression increases expression of many lysosomal genes even in TFEB-depleted cells

  • This indicates non-redundant functions of these related transcription factors

• Functional differences in TFE3 fusion proteins:

  • TFE3-splicing factor fusions drive transformation of kidney cells and promote distinct oncogenic phenotypes

  • Different fusion partners lead to differential alterations in the transcriptome and RNA splicing landscape

  • This partner-dependent functionality explains the heterogeneity observed in TFE3 fusion-positive tumors