FHL2 Antibody

What is FHL2 and why is it important in research?

FHL2 (also known as SLIM3 and DRAL) is a 30-32 kDa member of the four-and-a-half LIM domain-only protein family. It shows tissue-specific expression patterns in myocardium, skeletal muscle, and prostatic epithelium. FHL2 is particularly notable for its strong transactivation of the androgen receptor (AR) . The protein contains four distinct LIM domains (amino acids 40-92, 101-153, 162-212, and 221-275) that all bind to the AR . FHL2 plays critical roles in regulating cell proliferation, survival, adhesion, motility, and signal transduction in a cell type and tissue-dependent manner . Its involvement in immune function, specifically in spleen T cell-dependent B cell activation and antibody response, makes it an important target for immunological research .

What are the common applications for FHL2 antibodies in research?

FHL2 antibodies are versatile tools employed in multiple research applications:

• Western blotting: Detecting FHL2 protein expression in cell lysates (e.g., HT1080 human fibrosarcoma and MG-63 human osteosarcoma cell lines)

• Immunohistochemistry/Immunocytochemistry: Analyzing FHL2 tissue distribution and subcellular localization

• Immunofluorescence: Examining dynamic changes in FHL2 localization

• Chromatin immunoprecipitation (ChIP): Investigating FHL2's role in transcriptional regulation and its association with specific promoters, such as TGF-β1

• Co-immunoprecipitation: Studying protein-protein interactions involving FHL2

How should FHL2 antibodies be stored and handled for optimal results?

For optimal antibody performance and longevity:

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles

• Store at -20 to -70°C for up to 12 months from date of receipt as supplied

• After reconstitution, antibody can be stored at 2 to 8°C under sterile conditions for 1 month

• For longer storage after reconstitution, keep at -20 to -70°C under sterile conditions for up to 6 months

• During experimental use, maintain antibodies on ice and avoid extended exposure to room temperature

What tissues or cell types commonly express FHL2?

FHL2 exhibits a tissue-specific expression pattern that researchers should consider when designing experiments:

• Highest expression in myocardium (heart muscle)

• Strong expression in skeletal muscle

• Notable expression in prostatic epithelium

• Significant expression in different regions of the spleen, including white and red pulp, and in splenic B cells

• Expression has been detected in liver tissue

• Various cell lines including HT1080 (human fibrosarcoma), MG-63 (human osteosarcoma), NIH-3T3 (mouse fibroblasts), and NBT-II (rat cell line) show detectable FHL2 expression

How can I effectively validate FHL2 antibody specificity for my research?

Comprehensive antibody validation requires multiple complementary approaches:

• Orthogonal validation: Compare protein expression data from antibody-based methods with orthogonal techniques such as mass spectrometry or RNA expression analysis.

• Independent antibody validation: Compare results using multiple antibodies targeting different epitopes of FHL2. For example, comparing staining patterns of NBP1-84978 and NBP1-84979 across human cerebellum, heart muscle, liver, and skeletal muscle can verify consistent protein distribution patterns .

• Genetic approaches: Utilize FHL2 knockout models (FHL2^-/-^ mice or cells) as negative controls in your experiments. These models provide stringent specificity controls, as demonstrated in ChIP-qPCR assays where FHL2^-/-^ mouse embryonic fibroblasts served as negative controls .

• Recombinant protein competition: Pre-incubate your antibody with recombinant FHL2 protein before performing detection experiments to confirm binding specificity.

• Western blot analysis: Verify that your antibody detects a single band of the expected molecular weight (approximately 32 kDa for FHL2) .

What are the considerations for using FHL2 antibody in ChIP assays?

When performing chromatin immunoprecipitation with FHL2 antibodies:

• Experimental design: Since FHL2 lacks a DNA-binding domain, it associates with promoters through interactions with transcription factors. Design your ChIP-qPCR primers to target regions containing binding sites for known FHL2-interacting factors such as AP-1, NF-κB, and androgen receptor .

• Controls: Include:

  • Negative control antibody (IgG from the same species)

  • FHL2 knockout or knockdown cells as biological negative controls

  • Positive control regions where FHL2 binding is established (e.g., TGF-β1 promoter)

  • Input chromatin samples

• Cross-linking conditions: Standard formaldehyde cross-linking has been successfully employed for FHL2 ChIP experiments .

• Target validation: Multiple primer pairs targeting different regions of the promoter of interest should be used to confirm binding specificity, as was done for TGF-β1 promoter analysis .

• Comparative analysis: Consider parallel ChIP experiments with antibodies against known FHL2-interacting transcription factors like c-Jun and c-Fos (AP-1 complex components) to establish co-occupancy .

How does subcellular localization of FHL2 influence experimental design and interpretation?

FHL2 exhibits dynamic subcellular localization that is cell-type and context-dependent:

• Cell cycle dependence: The cellular localization of FHL2 in granulosa cell tumor (GCT) cells is cell cycle dependent . This necessitates cell synchronization or cell cycle analysis when studying FHL2 localization.

• Nuclear vs. cytoplasmic functions: FHL2 can function in both compartments:

  • Nuclear FHL2 acts as a transcriptional co-regulator for various transcription factors, including NF-κB and AP-1

  • Cytoplasmic FHL2 may interact with cytoskeletal components and signaling molecules

• Experimental implications:

  • Include subcellular fractionation controls in Western blot analyses

  • Utilize co-staining with compartment-specific markers in immunofluorescence studies

  • Consider dual fixation methods to preserve both cytoplasmic and nuclear localization patterns

  • Interpret results in the context of known FHL2 shuttling mechanisms and regulatory pathways

What methodological approaches can resolve contradictory findings in FHL2 functional studies?

Contradictory findings regarding FHL2 function may arise due to its context-dependent activities:

• Cell/tissue type considerations: FHL2 functions in a cell type and tissue-dependent manner . Experiments should be performed in multiple relevant cell lines and validated in primary cells when possible.

• Expression level impact: Both knockdown and overexpression studies should be conducted, as FHL2 may exert different effects depending on expression levels. For example:

  • Knockdown of FHL2 suppresses GCT cell growth and reduces viability

  • Ectopic expression promotes cell growth and enhances viability

• Pathway analysis integration:

  • Examine multiple downstream pathways simultaneously (e.g., AKT1 signaling, TGF-β1 regulation)

  • Use phospho-specific antibodies to differentiate between total protein levels and activated signaling components

• Temporal dynamics: Implement time-course experiments to distinguish between immediate and delayed effects of FHL2 modulation.

• In vivo validation: Confirm in vitro findings with appropriate animal models, as demonstrated with FHL2's role in GCT progression .

How can FHL2 antibodies be employed to investigate immune response mechanisms?

FHL2 plays crucial roles in immune function, particularly in splenic B cell responses:

• Germinal center reaction analysis:

  • Use FHL2 antibodies in combination with B cell markers to study germinal center structure and dark zone/light zone (DZ/LZ) distribution

  • Flow cytometry applications can quantify B cell subtypes in FHL2-sufficient versus FHL2-deficient models

• Class-switch recombination (CSR) investigation:

  • Combine FHL2 antibody staining with activation-induced cytidine deaminase (AID) detection to examine CSR mechanisms

  • Study the relationship between FHL2 expression and IgG1 production in response to T cell-dependent antigens like SRBC

• Plasma cell differentiation:

  • Track FHL2's influence on B cell to plasma cell transition using appropriate cell surface markers

  • Correlate FHL2 expression with antibody production capacity

• Cytokine production analysis:

  • Investigate FHL2's role in regulating CXCL12 and CXCL13 production in the spleen microenvironment

  • Examine how FHL2 influences the expression of cytokines critical for germinal center formation and maintenance

What strategies are effective for studying FHL2's role in cancer progression using antibody-based techniques?

FHL2 is implicated in cancer development, particularly in ovarian granulosa cell tumors (GCTs):

• Expression profiling:

  • Use immunohistochemistry to compare FHL2 expression between normal tissues and tumor samples

  • Quantify expression levels through image analysis software for correlation with clinical outcomes

• Functional studies:

  • Combine FHL2 knockdown/overexpression with antibody detection of downstream targets like AKT1

  • Investigate how FHL2 modulation affects cancer cell growth, viability, and migration in vitro

• Mechanistic investigations:

  • Utilize co-immunoprecipitation with FHL2 antibodies to identify interaction partners in cancer cells

  • Perform ChIP-qPCR to examine FHL2's association with promoters of oncogenes or tumor suppressors

• Therapeutic target assessment:

  • Evaluate changes in FHL2 expression or localization in response to various anti-cancer treatments

  • Develop techniques to monitor FHL2-dependent pathways as potential biomarkers for treatment response

How should experiments be designed to investigate FHL2's transcriptional regulatory functions?

FHL2 functions as a transcriptional co-regulator without direct DNA-binding capability:

• Transcription factor interaction studies:

  • Use co-immunoprecipitation with FHL2 antibodies to identify associated transcription factors (e.g., NF-κB, AP-1)

  • Perform sequential ChIP (re-ChIP) to confirm co-occupancy of FHL2 with specific transcription factors on target promoters

• Promoter analysis workflows:

  • Identify potential FHL2-regulated genes through expression profiling in FHL2 knockdown/knockout models

  • Confirm direct regulation through ChIP-qPCR targeting promoter regions of candidate genes

  • Utilize reporter gene assays with wild-type and mutated promoter constructs to validate functional significance

• Integrative approaches:

  • Combine ChIP-seq, RNA-seq, and protein-protein interaction data to construct comprehensive FHL2 regulatory networks

  • Validate key nodes through targeted experiments with FHL2 antibodies

• Technical considerations:

  • When designing ChIP-qPCR primers, target regions containing binding sites for known FHL2-interacting transcription factors

  • Include RNA polymerase II ChIP as a positive control for active transcription

  • Compare chromatin architecture between wild-type and FHL2-deficient cells to understand regulatory mechanisms

What are common challenges in Western blot detection of FHL2 and how can they be overcome?

Researchers may encounter several challenges when detecting FHL2 by Western blot:

• Multiple bands or unexpected molecular weight:

  • FHL2 has potential alternate start sites (Met115 and Met110) and splice variants that can affect band pattern

  • Ensure proper sample preparation to prevent protein degradation (use protease inhibitors)

  • Optimize antibody concentration (recommended range: 0.04-0.4 μg/ml)

  • Try different reducing conditions (FHL2 detection works well under reducing conditions using Immunoblot Buffer Group 8)

• Weak signal:

  • Consider tissue/cell-specific expression levels; FHL2 shows tissue-specific patterns

  • Use PVDF membrane which has been successfully employed for FHL2 detection

  • Optimize blocking conditions and incubation times

  • Ensure appropriate secondary antibody selection (e.g., HRP-conjugated Anti-Goat IgG for goat primary antibodies)

• Background issues:

  • Increase washing duration and frequency

  • Test different blocking agents (BSA vs. non-fat dry milk)

  • Reduce primary and secondary antibody concentrations

  • For polyclonal antibodies, consider pre-absorption with non-specific proteins

How can immunohistochemistry protocols be optimized for FHL2 detection in different tissue types?

Optimizing IHC protocols for FHL2 detection requires tissue-specific considerations:

• Antigen retrieval:

  • For paraffin-embedded tissues, HIER (Heat-Induced Epitope Retrieval) at pH 6 is recommended

  • Optimize retrieval time based on tissue type and fixation method

• Antibody dilution optimization:

  • Start with recommended dilution range (1:200-1:500 for paraffin sections)

  • Perform titration experiments to determine optimal concentration for each tissue type

  • Consider longer incubation times at lower antibody concentrations to enhance specific staining

• Tissue-specific considerations:

  • For tissues with high endogenous FHL2 expression (heart, skeletal muscle), lower antibody concentrations may be sufficient

  • For tissues with lower expression, signal amplification systems may be beneficial

  • Use appropriate positive and negative control tissues in each experiment

• Signal detection systems:

  • Compare different detection methods (DAB, AEC, fluorescent secondary antibodies)

  • For co-localization studies, select compatible fluorophores with minimal spectral overlap

  • Consider tyramide signal amplification for detecting low-abundance targets

What factors contribute to variability in FHL2 antibody performance across different experimental systems?

Several factors can affect FHL2 antibody performance across experimental systems:

How should researchers interpret changes in FHL2 expression patterns in disease models?

Interpreting FHL2 expression changes requires careful consideration of multiple factors:

• Baseline expression reference:

  • Establish normal expression patterns in relevant tissues before assessing disease-related changes

  • FHL2 normally shows tissue-specific expression in myocardium, skeletal muscle, and prostatic epithelium

• Subcellular localization analysis:

  • Assess not only total expression levels but also subcellular distribution patterns

  • FHL2 cellular localization can be cell cycle dependent

  • Nuclear translocation may indicate activation of specific transcriptional programs

• Context-dependent function:

  • FHL2 can act as both positive and negative regulator depending on context

  • It functions as a negative regulator of TGF-β1

  • Overexpression in GCTs promotes tumor progression

• Correlation with clinical parameters:

  • Relate FHL2 expression changes to disease progression, treatment response, or patient outcomes

  • Consider creating quantitative scoring systems for FHL2 immunohistochemistry

What comparative approaches can distinguish between FHL2-specific effects and non-specific observations?

To ensure observed effects are truly FHL2-specific:

• Genetic validation techniques:

  • Use multiple independent FHL2 knockdown/knockout models

  • Perform rescue experiments with wild-type FHL2 re-expression

  • As demonstrated in GCT research, ectopic expression of FHL2 should reverse knockdown phenotypes

• Pathway reconstruction:

  • If FHL2 regulates a pathway (e.g., AKT1 signaling), manipulating downstream components should phenocopy FHL2 modulation

  • Constitutively active AKT1 rescued FHL2 knockdown-induced arrest of GCT cell growth

• Multiple antibody approach:

  • Use different antibodies targeting distinct FHL2 epitopes to confirm findings

  • Compare results from antibody-based detection with other methodologies

• Cross-species validation:

  • Given the high conservation of FHL2 across species, key findings should be reproducible across human, mouse, and rat systems

  • Compare results between different model organisms while accounting for species-specific functions

What experimental design considerations are essential when using FHL2 antibodies in complex immunological studies?

When investigating FHL2's role in immune responses:

• Cell-intrinsic versus microenvironment effects:

  • FHL2 defects in B cell responses may not be B cell intrinsic but related to the spleen microenvironment

  • Design bone marrow transplantation experiments to distinguish cell-autonomous effects

  • B cell-deficient μMT mice transplanted with wild-type or FHL2^-/-^ bone marrow can help identify cell-specific roles

• Immune challenge models:

  • Select appropriate immune challenges (e.g., SRBC for T cell-dependent responses)

  • Monitor multiple parameters including:

    • Germinal center formation and structure

    • Class-switch recombination efficiency

    • Antibody production (type and quantity)

    • Plasma cell generation

• Cytokine analysis integration:

  • Measure cytokines regulated by FHL2 (CXCL12, CXCL13) that impact immune cell trafficking and positioning

  • Use multiplex cytokine assays or single-cell techniques to capture the complexity of immune responses

• Comprehensive immune phenotyping:

  • Assess multiple immune cell populations simultaneously

  • Examine both basal state and activation-induced changes in FHL2 expression and function

  • Consider the impact of FHL2 on immune cell migration, positioning, and intercellular communication