FHL2 Antibody

What is FHL2 protein and what are its alternative nomenclatures?

FHL2 (Four and a half LIM domains protein 2) is also known as DRAL and SLIM3 . It belongs to the four-and-a half class of the LIM domain-only protein family . FHL2 is 279 amino acids in length in humans and contains four distinct LIM domains (at amino acid positions 40-92, 101-153, 162-212, and 221-275) . The protein functions primarily as an adaptor or scaffold protein, capable of modulating various signaling pathways involved in diverse cellular functions . FHL2 is considered the best-studied member of the FHL protein family .

What is the molecular weight of FHL2 and how is it typically detected?

FHL2 has a molecular weight of approximately 32-33 kDa as detected by Western blotting . When using mouse anti-FHL2 antibody (clone AB04-4H8), a band of approximately 33 kDa is detected in HeLa cell lysates . Similarly, using Goat Anti-Human FHL2 Antigen Affinity-purified Polyclonal Antibody, a specific band for FHL2 is detected at approximately 32 kDa in lysates from HT1080 human fibrosarcoma and MG-63 human osteosarcoma cell lines . For optimal detection, Western blots should be conducted under reducing conditions with appropriate immunoblot buffer systems .

In which tissues is FHL2 predominantly expressed?

FHL2 exhibits a tissue-specific expression pattern. It is prominently expressed in the myocardium of the heart, skeletal muscle, and the epithelial cells of the prostate . In the prostate, FHL2 colocalizes with the androgen receptor (AR) in the nucleus . Immunogold labeling studies have also identified FHL2 in cardiac myofibers, with the highest density of immunogold particles observed in the middle of the I-band, suggesting that most cytosolic FHL2 is bound to the spring region of titin . This restricted expression pattern is significant for understanding its tissue-specific functions.

What are the recommended protocols for detecting FHL2 translocation between cytoplasm and nucleus?

To study FHL2 translocation, researchers should employ immunofluorescence analysis under different cellular conditions. Based on established protocols, cells should be transfected with a tagged FHL2 expression vector (such as Myc-FHL2) and subjected to different treatments . Under serum deprivation conditions, FHL2 primarily localizes to the cytoplasm (observed in approximately 80% of cells), particularly at focal adhesion complexes . Stimulation with 20% serum or UV light induces significant translocation of FHL2 into the nucleus within 3 hours .

To definitively determine whether cytoplasmic localization is due to sequestration or nuclear exclusion, treat cells with leptomycin B (an inhibitor of active nuclear export) . After leptomycin B treatment, approximately 45% of cells will show FHL2-positive signal in both nucleus and cytoplasm, compared to 20% under basal conditions, indicating that active nuclear export (Crm1/exportin-dependent) is partly responsible for cytoplasmic localization of FHL2 . Confocal laser microscopy is recommended for precise localization analysis.

How should I optimize Western blotting for FHL2 detection?

For optimal Western blot detection of FHL2, follow these methodological guidelines:

• Use PVDF membrane for protein transfer

• Apply 1 μg/mL of anti-FHL2 antibody (such as Goat Anti-Human FHL2 Antigen Affinity-purified Polyclonal Antibody)

• Follow with an appropriate HRP-conjugated secondary antibody

• Conduct the experiment under reducing conditions

• Use appropriate immunoblot buffer systems (e.g., Immunoblot Buffer Group 8 has been validated)

When properly executed, you should detect a specific band at approximately 32-33 kDa. Cell lines known to express detectable levels of FHL2 include HeLa (human cervical cancer), HT1080 (human fibrosarcoma), and MG-63 (human osteosarcoma) .

What methods are effective for studying FHL2 in immune response contexts?

To investigate FHL2's role in immune responses, particularly in germinal center reactions and B cell maturation, researchers should consider these methodological approaches:

• Animal models: Compare wild-type (WT) mice with FHL2-/- knockout mice to assess phenotypic differences in immune structures and responses

• T cell-dependent immune response induction: Elicit responses using SRBCs (sheep red blood cells) and measure germinal center area changes

• Histological analysis: Examine spleen follicle size and B cell populations

• Molecular analysis: Measure expression of relevant markers including IgG1, activation-induced cytidine deaminase (AID) mRNA

• Bone marrow transplantation: Use B cell-deficient μMT mice transplanted with WT or FHL2-/- bone marrow to determine if immune response defects are B cell intrinsic

• Microenvironment analysis: Analyze spleen lysates for chemokine levels (particularly CXCL12 and CXCL13)

These approaches have revealed that FHL2 expression is essential for normal germinal center reactions and proper induction of class-switch recombination in response to T cell-dependent antigens .

How does FHL2 function as a transcriptional coactivator?

FHL2 functions as a tissue-specific coactivator of the androgen receptor (AR) through direct protein interactions. Unlike many nuclear receptor cofactors, FHL2 binds to the surface generated by the interaction of the holo-ligand binding domain (LBD) together with the N-terminus of the AR . The interaction is specific to AR among nuclear receptors .

Mechanistically, FHL2 contains a strong, autonomous transactivation function . Its coactivator function is particularly relevant in tissues where it is highly expressed, such as prostatic epithelium, where it colocalizes with AR in the nucleus . To study this coactivator function, researchers should employ modified yeast two-hybrid systems using a human AR in which the DNA-binding domain is replaced by the Gal4 DNA-binding domain . This approach allows identification of cofactors that bind to the complete receptor structure rather than isolated domains.

What is the role of FHL2 in cancer progression?

FHL2's role in cancer progression is complex and tissue-dependent. Expression patterns of FHL2 vary significantly between different cancer types, with FHL2 acting as either a tumor suppressor or an oncogene depending on the tissue context . This dual functionality makes it a particularly interesting target for cancer research.

The molecular mechanisms underlying these contradictory roles involve FHL2's ability to interact with various signaling pathways through its LIM domains . These domains allow FHL2 to interact with a broad range of unrelated molecules , potentially explaining its diverse effects in different cellular contexts.

When investigating FHL2 in cancer models, researchers should:

• Compare expression levels across multiple cancer types

• Correlate expression with clinical outcomes

• Analyze pathway interactions specific to each cancer type

• Consider the tissue of origin and its normal FHL2 expression pattern

What is known about FHL2's role in cardiac function and pathology?

FHL2 plays a significant role in cardiac function, and its dysregulation is associated with cardiac pathologies:

• Expression pattern: FHL2 is normally highly expressed in the myocardium

• Subcellular localization: Immunogold labeling reveals that FHL2 is predominantly localized in the middle of the I-band in cardiac myofibers, suggesting association with the spring region of titin

• Pathological changes: FHL2 is downregulated in hypertrophic cardiomyopathy (HCM)

• Quantitative changes: Abundance of gold particles is approximately 50% lower in HCM and homozygous knock-in cardiac sections compared to donor tissue

• Protective effects: Both wild-type FHL2 and variants partially protected against phenylephrine- or endothelin-1-induced hypertrophic responses

These findings suggest that FHL2 may have cardioprotective functions and that its downregulation could contribute to the development of cardiac hypertrophy. For studying FHL2 in cardiac contexts, immunogold labeling of ultrathin sections of ventricular tissue is recommended for precise localization analysis .

Why might I observe different subcellular localization patterns for FHL2?

Differential subcellular localization of FHL2 is primarily regulated by:

• Cellular stimulation status: Under serum deprivation, FHL2 is predominantly cytoplasmic (80% of cells), while serum stimulation or UV light exposure induces nuclear translocation within 3 hours

• Active nuclear export: FHL2 is subject to Crm1/exportin-dependent nuclear export, as demonstrated by leptomycin B treatment studies

• Cell type differences: FHL2 localization may vary between cell types based on their expression of interaction partners

• Experimental artifacts: Fixation methods and antibody specificity can affect apparent localization

If observing inconsistent localization patterns, verify:

• Serum conditions are consistent across experiments

• Cells are fixed at appropriate time points after stimulation

• Antibody specificity using appropriate controls

• Use confocal microscopy for precise localization determination

How should I validate antibody specificity when working with FHL2?

To ensure antibody specificity when studying FHL2:

• Validate molecular weight: Confirm detection of a band at approximately 32-33 kDa by Western blot

• Include positive controls: Use cell lines known to express FHL2 (HeLa, HT1080, MG-63)

• Include negative controls: Use FHL2-/- samples or knockdown cells when available

• Verify tissue specificity: Confirm expression in tissues known to express FHL2 (myocardium, skeletal muscle, prostatic epithelium)

• Cross-reactivity testing: Test for cross-reactivity with other FHL family members

• Test multiple antibodies: Compare results using antibodies targeting different epitopes (e.g., N-terminal vs. C-terminal)

Remember that optimal antibody dilutions should be determined by each laboratory for each application, as indicated in manufacturer guidelines .

What factors might explain contradictory results between in vitro and in vivo FHL2 studies?

Discrepancies between in vitro and in vivo FHL2 studies may be attributed to:

• Microenvironment factors: In vivo studies reveal that FHL2-/- mice have disturbed spleen microenvironments with reduced CXCL12 and CXCL13 levels compared to wild type

• Cell-extrinsic effects: While FHL2-/- B cells can undergo class-switch recombination in vitro, FHL2-/- mice show defects in T cell-dependent B cell responses in vivo

• Tissue-specific interactions: FHL2 interacts with tissue-specific factors that may be absent in vitro

• Signal integration: FHL2 functions as an adaptor protein integrating multiple signals that may be incompletely represented in simplified in vitro systems

• Dynamic regulation: FHL2 exhibits dynamic regulation (e.g., serum-induced expression and nuclear translocation) that may be difficult to model in vitro

To reconcile such discrepancies, consider combination approaches:

• Compare isolated cell behavior with tissue explants

• Use conditional knockout models for tissue-specific deletion

• Employ transplantation studies (e.g., bone marrow transplantation into μMT mice)

• Analyze microenvironmental factors that might influence FHL2 function

What are promising applications for targeting FHL2 in therapeutic contexts?

Based on current understanding of FHL2 biology, several therapeutic applications warrant investigation:

• Cancer therapy: Given FHL2's tissue-specific oncogenic or tumor-suppressive roles , developing targeted approaches for specific cancer types may be valuable

• Immune modulation: FHL2's role in germinal center reactions and B cell responses suggests potential for modulating antibody-mediated immune disorders

• Cardiac protection: FHL2's downregulation in hypertrophic cardiomyopathy indicates potential cardioprotective applications

• Androgen signaling modulation: As a coactivator of the androgen receptor , FHL2 may represent a target for prostate disorders

Research should focus on developing tools for tissue-specific modulation of FHL2 function rather than global inhibition, given its diverse roles across tissues.

What experimental approaches might reveal new FHL2 functions?

To uncover novel FHL2 functions, consider these experimental approaches:

• Interactome analysis: Comprehensive mapping of tissue-specific FHL2 interaction partners

• Domain-specific mutagenesis: Systematic mutation of individual LIM domains to dissect their contributions to different functions

• Conditional knockout models: Tissue-specific and inducible deletion to bypass developmental effects

• Transcriptomics in FHL2-manipulated systems: RNA-seq analysis of FHL2 knockout or overexpression models

• Phosphoproteomics: Identification of signaling pathways altered by FHL2 manipulation

• Super-resolution microscopy: Detailed analysis of FHL2 subcellular localization and dynamics

• Single-cell approaches: Analysis of cell-to-cell variability in FHL2 expression and function

These approaches may reveal additional roles beyond the currently established functions in transcriptional regulation, signal transduction, and tissue-specific development.