FOSL2 Antibody

What is FOSL2 and why is it significant in research?

FOSL2 (FOS-like antigen 2) is a basic region-leucine zipper motif transcription factor that belongs to the AP-1 transcription factor family. It is widely expressed in mammalian tissues and plays crucial roles in various physiological and pathological processes . The calculated molecular weight of FOSL2 is approximately 35.2 kDa (326 amino acids), though the observed molecular weight in Western blot applications is often reported as approximately 31 kDa .

FOSL2 has been implicated in:

• Bone development and photoperiodic regulation

• Cancer progression, particularly in non-small cell lung cancer

• Fibrotic diseases affecting multiple organs including kidney, skin, and lungs

• Immune cell differentiation and function, particularly in Th17 cells

The clinical and research significance of FOSL2 stems from its involvement in these diverse pathophysiological processes, making it an important target for understanding disease mechanisms.

How do I select the appropriate FOSL2 antibody for my research?

Selection of the optimal FOSL2 antibody depends on several factors:

For critical experiments, consider using antibodies that have been validated in publications with experimental conditions similar to yours. The search results indicate that antibodies like 15832-1-AP have been validated in multiple publications across different applications .

What are the optimal experimental conditions for Western blot detection of FOSL2?

For reliable Western blot detection of FOSL2:

Sample preparation:

• Use cell lines with known FOSL2 expression (MCF-7, Jurkat, PC-3 cells work well as positive controls)

• Add protease inhibitors to prevent degradation

• For stimulation experiments, TGF-β1 treatment increases FOSL2 expression (peaks at 72h in A549 cells)

Protocol optimization:

• Recommended dilution range: 1:200-1:1000 for most FOSL2 antibodies

• Expected band: ~31-35 kDa

• Sample-dependent optimization is required; titrate antibodies in each testing system

Controls to include:

• Positive control: Transfected cells overexpressing FOSL2

• Negative control: FOSL2 knockdown samples via siRNA

• Loading control: GAPDH or other standard housekeeping proteins

How can I effectively use FOSL2 antibodies for immunohistochemistry?

For optimal immunohistochemistry (IHC) with FOSL2 antibodies:

Tissue preparation:

• Formalin-fixed paraffin-embedded (FFPE) sections are commonly used

• Antigen retrieval is critical: H2O2 treatment (e.g., 3% for 10 minutes) helps eliminate endogenous peroxidase activity

Staining protocol:

• Primary antibody dilution: 1:100-1:200 for most FOSL2 antibodies

• Incubation: Overnight at 4°C yields optimal results

• Secondary antibody: HRP-conjugated anti-rabbit/mouse IgG (depending on primary host)

• Visualization: DAB (3,3'-diaminobenzidine) with hematoxylin counterstain

Analysis considerations:

• Nuclear localization is expected for FOSL2 as a transcription factor

• In disease states like fibrosis, increased expression may be observed

• Quantify expression using appropriate imaging software for intensity and distribution

How can I use FOSL2 antibodies to study its role in TGF-β1 signaling and fibrosis?

TGF-β1 signaling is a key pathway in which FOSL2 plays a regulatory role, particularly in fibrotic diseases:

Experimental design for studying FOSL2 in TGF-β1 pathway:

• Expression analysis:

  • Treat cells with TGF-β1 (2 ng/ml is standard) and monitor FOSL2 expression over time (24h-72h)

  • Western blot analysis will show FOSL2 levels increase substantially, with maximal expression at 72h (approximately 4× basal level)

• Functional studies:

  • Use siRNA to knockdown FOSL2 (Lipofectamine RNAiMAX recommended for transfection)

  • Assess impact on TGF-β1-induced migration using Transwell migration assays

  • Evaluate effects on EMT markers (E-cadherin, fibronectin, α-SMA)

• Mechanistic investigations:

  • Examine FOSL2's interaction with Smad3 using co-immunoprecipitation

  • Assess P300 binding to Smad3 and acetylation status of Smad3

  • Use reporter assays (e.g., 3TP-lux reporter) to measure TGF-β response elements

What approaches can be used to study FOSL2's function in transcriptional regulation?

As a transcription factor, FOSL2's primary function is regulating gene expression:

ChIP-based approaches:

• Use ChIP-certified FOSL2 antibodies for optimal results

• Equal amounts of two FOSL2 antisera directed against different epitopes can improve ChIP efficiency

• Include appropriate controls: RNA polymerase II (positive control) and IgG (negative control)

Target gene identification:

• ChIP-seq analysis can reveal genome-wide binding patterns

• Bioinformatic analysis of validated sets of FOSL2-regulated and non-regulated genes reveals that the FOSL2 regulon is limited by genomic target selection rules

• For validation, perform qPCR on specific targets identified in ChIP-seq experiments

Functional validation:

• Combine with knockdown/overexpression studies to confirm regulatory relationships

• Analysis of promoter regions with conserved FOSL2 binding motifs (e.g., tgcgtca sites)

How do I investigate the differential roles of FOSL1, FOSL2, and BATF in immune cell differentiation?

The interplay between these AP-1 transcription factors is critical in immune cell development, particularly in Th17 differentiation:

Comparative analysis approach:

• FOSL1 and FOSL2 co-repress Th17 fate-specification, whereas BATF promotes the Th17 lineage

• Species-specific differences exist: FOSL1 plays different roles in human versus mouse systems

Experimental strategy:

• Genomic binding analysis:

  • ChIP-seq reveals that FOSL1, FOSL2, and BATF share occupancy over regulatory regions of genes involved in Th17 lineage commitment

  • Compare binding profiles to identify unique and overlapping targets

• Protein interaction studies:

  • These AP-1 factors share protein interacting partners, suggesting mechanisms for functional interplay

  • Use co-immunoprecipitation or proximity ligation assays to map interaction networks

• Disease relevance:

  • Genomic binding sites harbor hundreds of autoimmune disease-linked SNPs

  • Functional studies can demonstrate how these SNPs alter transcription factor binding and affect gene expression

What are effective approaches for studying FOSL2's role in renal fibrosis?

Based on recent findings, FOSL2 plays an essential role in promoting renal fibrosis:

In vivo approaches:

• Unilateral ureteral obstruction (UUO) mouse model shows increased FOSL2 expression

• FOSL2 knockdown via adenovirus-delivered shRNA (target site: 5'-GGACCUGCAGUGGAUGGUACA-3')

• Administration: Tail vein injection once a week, with UUO performed 2 days after first injection

• Assessment: Analyze kidneys at 14 days post-UUO surgery

Mechanistic investigation:

• Focus on SGK1 (serum/glucocorticoid regulated kinase 1) as a downstream mediator

• FOSL2 binds to the SGK1 promoter and enhances its expression

• SGK1 overexpression reverses effects of FOSL2 silencing in TGF-β1-induced cells

Analysis parameters:

• Examine interstitial fibrosis, extracellular matrix deposition, and EMT markers

• Key targets: fibronectin, α-SMA, collagen type I (Col1a1 and Col1a2), Col5a1, and E-cadherin

How do I troubleshoot inconsistent results when using FOSL2 antibodies?

When facing challenges with FOSL2 antibody applications:

Common issues and solutions:

Validation strategies:

• Compare results using antibodies from different sources or targeting different epitopes

• Include appropriate positive controls (e.g., cell lines: MCF-7, Jurkat, PC-3)

• Confirm specificity using FOSL2 knockdown samples

How can FOSL2 antibodies be used to explore its role in disease biomarker development?

FOSL2's involvement in multiple pathological processes suggests potential as a disease biomarker:

Cancer applications:

• FOSL2 facilitates TGF-β1-induced migration in non-small cell lung cancer

• FOSL2 expression correlates with activated Smad3 expression in clinical NSCLC samples

• Antibody-based tissue microarray analysis can evaluate correlation with clinical outcomes

Fibrotic disease monitoring:

• FOSL2/SGK1 axis represents a potential biomarker for renal fibrosis progression

• IHC analysis of biopsy samples could assess FOSL2 expression as a predictive marker

Methodological considerations:

• Standardized protocols and validated antibodies are essential for biomarker development

• Multiple antibody validation across diverse patient cohorts required for clinical applications

• Consider multiplexed approaches combining FOSL2 with other markers for improved specificity