FMNL1 Antibody

Introduction to FMNL1 and FMNL1 Antibodies

Formin-like 1 (FMNL1) is a protein that belongs to the formin homology family, characterized by unique structural domains including a diaphanous autoregulatory domain, a formin homology 2 domain, and a Rho GTPase-binding/formin homology 3 domain . FMNL1 plays crucial roles in the regulation of cell morphology, cytoskeletal organization, and is required for cortical actin filament dynamics and cell shape maintenance . This protein is encoded by the FMNL1 gene in humans, with several aliases including C17orf1B, FMNL, and FRL1 .

FMNL1 antibodies are specialized immunoglobulins designed to specifically bind to and detect FMNL1 protein in various experimental applications. These antibodies serve as important tools for investigating FMNL1 expression, localization, and function in both normal and pathological conditions. The development of reliable FMNL1 antibodies has significantly advanced our understanding of this protein's role in cellular processes and disease mechanisms.

Types and Characteristics of FMNL1 Antibodies

FMNL1 antibodies are available in various formats, each with specific characteristics suited for different research applications. They can be broadly categorized into monoclonal and polyclonal antibodies.

Monoclonal FMNL1 Antibodies

Monoclonal antibodies are derived from a single B-cell clone, ensuring high specificity and consistency between batches. A notable example is the FMNL1 (A-4) antibody developed by Santa Cruz Biotechnology, which is a mouse monoclonal IgG1 kappa light chain antibody that detects FMNL1 protein of mouse, rat, and human origin . This antibody is available in multiple formats:

• Non-conjugated (sc-390466)

• Agarose-conjugated (sc-390466 AC) for immunoprecipitation

• Horseradish peroxidase-conjugated (sc-390466 HRP) for enhanced detection sensitivity

• Various fluorescent conjugates including phycoerythrin (PE), fluorescein isothiocyanate (FITC), and Alexa Fluor® variants

Polyclonal FMNL1 Antibodies

Polyclonal antibodies are derived from multiple B-cell clones and recognize different epitopes on the FMNL1 protein. Examples include:

• Invitrogen's rabbit polyclonal antibodies (PA5-34477 and PA5-88912) with reactivity to human, mouse, and rat FMNL1

• Abcam's rabbit polyclonal antibody (ab224247) generated against a recombinant fragment within human FMNL1 (amino acids 850-1000)

• The Human Protein Atlas (HPA) antibody, designed for formalin-fixed paraffin-embedded (FFPE) tissue analysis, targeting a specific FMNL1 sequence (amino acids 880-991)

Table 1 provides a comprehensive comparison of commercially available FMNL1 antibodies:

Validation Methods for FMNL1 Antibodies

Rigorous validation is essential to ensure the specificity and reliability of FMNL1 antibodies. Several methods have been employed to validate these antibodies:

Antigen Array Testing

The HPA FMNL1 antibody was initially validated using an antigen array of 384 different Protein Epitope Signature Tags (PrESTs), where it exclusively interacted with its designated antigen . This testing confirmed the antibody's specificity for the FMNL1 protein and minimal cross-reactivity with other proteins.

Western Blot Validation

Western blotting has been extensively used to validate FMNL1 antibodies. The HPA antibody detected a single band of approximately 140 kDa in cell lines known to express FMNL1, including Jurkat (T-cell lymphoma), CCD18 (myofibroblast), MDA-MB-231 (breast cancer), Ovcar-4 and Hey (both ovarian cancer) . These findings correlate with the predicted molecular weights for FMNL1 isoforms (122-124 kDa), with the slightly higher observed weight being consistent with previous literature reports .

Transfection experiments provided further validation, where the antibody detected FMNL1 in cell lines that normally lack endogenous expression. After transfection with FMNL1 constructs, the antibody detected bands of expected sizes: 100 kDa for the C-terminal half of FMNL1 isoform 1 (FMNL1') and 130 kDa for the GFP-tagged version .

Immunofluorescence Validation

Immunofluorescence staining has confirmed the specificity of FMNL1 antibodies by demonstrating their ability to detect FMNL1 in transfected cells. The staining pattern observed with the HPA FMNL1 antibody matched the localization of GFP-tagged FMNL1 constructs, showing cytoplasmic and membrane localization, particularly at actin-rich protrusions .

Knockout Validation

The most stringent validation method involves testing antibodies in knockout systems where the target protein is absent. Although not explicitly detailed in the provided search results, knockout validation is mentioned for at least one FMNL1 antibody product , providing strong evidence for antibody specificity.

Applications of FMNL1 Antibodies in Research

FMNL1 antibodies have been employed in various experimental applications to investigate the expression, localization, and function of FMNL1 protein.

Western Blotting (WB)

Western blotting is widely used to detect and quantify FMNL1 protein in cell and tissue lysates. This application has revealed variable FMNL1 expression across different cell types, with particularly strong expression in immune cells such as T lymphocytes . Table 2 summarizes FMNL1 expression in various cell lines as detected by western blotting:

Immunohistochemistry (IHC)

FMNL1 antibodies suitable for immunohistochemistry on formalin-fixed paraffin-embedded (FFPE) tissues have been instrumental in characterizing FMNL1 expression across human tissues. The development of antibodies that work in routine diagnostic material has overcome a major hurdle in investigating FMNL1 expression in malignant diseases . Abcam's ab224247 antibody has been successfully used to detect FMNL1 in human lymph node and small intestine tissues .

Immunofluorescence (IF)

Immunofluorescence applications of FMNL1 antibodies have provided valuable insights into the subcellular localization of FMNL1 protein. Studies have shown that FMNL1 localizes to the cytoplasm and cell membranes, particularly at actin-rich protrusions . Co-localization studies with fluorescent phalloidin have revealed that FMNL1 associates with actin filaments, consistent with its role in actin dynamics .

Immunoprecipitation (IP) and ELISA

FMNL1 antibodies have been used in immunoprecipitation assays to isolate FMNL1 protein and its associated complexes, facilitating the study of protein-protein interactions . Enzyme-linked immunosorbent assay (ELISA) applications have enabled quantitative detection of FMNL1 protein and anti-FMNL1 autoantibodies, the latter being particularly relevant in the context of autoimmune diseases such as membranous nephropathy .

Tissue Expression Patterns

FMNL1 exhibits a distinctive expression pattern across human tissues. It is predominantly expressed in immune-related tissues including the spleen, lymph nodes, and bone marrow . Within these tissues, FMNL1 is highly expressed in various immune cells but shows low expression in other cell types such as tumor cells, hepatocyte-like cells, fibroblasts, and endothelial cells, as demonstrated by single-cell RNA sequencing (scRNA-seq) analysis .

The development of antibodies suitable for FFPE tissue analysis has enabled comprehensive characterization of FMNL1 expression across 28 human tissue types, revealing its enrichment in immune cells and potential overexpression in certain cancer types .

Functional Roles of FMNL1

FMNL1 plays several critical roles in cellular function:

1. Actin dynamics regulation: FMNL1 facilitates the nucleation and elongation of actin filaments, which are essential for cellular processes including motility and morphology .

2. Immune cell function: FMNL1 regulates macrophage survival and motility, contributing to immune responses .

3. Cell adhesion and migration: By inhibiting cell adhesion and migration, FMNL1 influences tissue homeostasis .

4. Apoptosis regulation: FMNL1 has been shown to block apoptotic cell death, potentially contributing to cell survival mechanisms .

FMNL1 interacts with several proteins important for actin dynamics and cellular signaling pathways, including Rac1, profilin 1, and profilin 2 .

FMNL1 in Cancer Research

FMNL1 has emerged as a significant biomarker in cancer research, with important implications for prognosis and treatment response:

FMNL1 in Autoimmune Disorders

Research has uncovered a potentially important role for FMNL1 in autoimmune disorders:

1. Anti-FMNL1 autoantibodies in membranous nephropathy: High-density peptide array screening identified anti-FMNL1 autoantibodies in sera of patients with membranous nephropathy (MN) .

2. Clinical significance: High levels of anti-FMNL1 IgG4 in MN patients were associated with lack of remission of proteinuria, suggesting a potential role in disease progression .

3. Histopathological findings: FMNL1-positive cells were detected in kidney tissues of MN patients, co-staining with CD68 (a macrophage marker) in glomeruli . This suggests that autoantibodies directed against cells involved in tissue repair, rather than just podocytes, might contribute to MN pathogenesis .

Future Perspectives in FMNL1 Antibody Research

FMNL1 antibody research continues to evolve, with several promising directions for future investigation:

1. Therapeutic applications: The development of therapeutic antibodies targeting FMNL1 may have potential in treating diseases where FMNL1 plays a pathogenic role.

2. Diagnostic tools: FMNL1 antibodies could serve as valuable diagnostic tools for identifying immune cell infiltration in tumors and predicting response to immunotherapy.

3. Mechanism elucidation: Further research using FMNL1 antibodies will help elucidate the mechanisms by which FMNL1+ immune cells contribute to inflammatory and anti-tumor responses.

4. Multiparameter analysis: Integration of FMNL1 antibodies into multiparameter imaging approaches, such as multiplexed quantitative immunofluorescence (mQIF), will provide deeper insights into cellular interactions within complex tissue microenvironments .