FAIM2 Antibody

What is FAIM2 and what are its key biological functions?

FAIM2, also known as Lifeguard (LFG), NMP35, or transmembrane BAX inhibitor motif containing 2 (TMBIM2), is a Fas antagonist primarily expressed in the nervous system. FAIM2 functions as an intrinsic neuroprotective factor that inhibits FAS-mediated apoptosis . It plays a crucial role in maintaining calcium balance in the endoplasmic reticulum, which contributes to its anti-apoptotic properties . FAIM2 is located in multiple cellular compartments including the Golgi apparatus, endoplasmic reticulum, and plasma membrane . Research has shown that FAIM2 directly interacts with the FAS receptor upstream of FAS-associated death domain containing protein (FADD), effectively blocking the apoptotic signaling cascade .

In neurons, FAIM2 increases resistance to FAS-mediated apoptosis, with FAIM2-deficient neurons showing greater susceptibility to combined oxygen-glucose deprivation in vitro and caspase-associated cell death after cerebral ischemia in vivo . Beyond the nervous system, FAIM2 anti-apoptotic activity has been implicated in various cancers, including non-small cell lung cancer, hepatocellular carcinoma, and breast carcinoma .

What should researchers know about FAIM2 antibody specifications for experimental planning?

When planning experiments using FAIM2 antibody, researchers should consider several key specifications:

Researchers should note that this antibody requires titration in each testing system to obtain optimal results, as performance can be sample-dependent . The discrepancy between calculated (35 kDa) and observed (30 kDa) molecular weights should be considered when interpreting results, as this may reflect post-translational modifications or protein processing in different tissue samples .

How should researchers optimize Western Blot protocols for FAIM2 detection?

For optimal Western Blot detection of FAIM2, researchers should follow these methodological steps:

• Sample preparation: FAIM2 is most reliably detected in brain tissue samples from mouse and rat models, as verified by previous studies . For other tissue types, additional validation steps are recommended.

• Protein loading and separation: Load 20-30 μg of total protein per lane and separate on 10-12% SDS-PAGE gels for optimal resolution around the 30 kDa mark where FAIM2 is observed.

• Antibody dilution optimization: Begin with a mid-range dilution (1:1000) of the FAIM2 antibody and adjust based on signal strength. A titration series (1:500, 1:1000, 1:2000) is recommended for first-time experiments to determine optimal concentration .

• Incubation conditions: Incubate primary antibody overnight at 4°C in blocking buffer containing 5% non-fat milk or BSA in TBST.

• Detection method: Use HRP-conjugated secondary antibodies and ECL detection systems for standard visualization, or consider fluorescent secondary antibodies for multiplexing with other proteins of interest.

• Positive controls: Include brain tissue lysates as positive controls, as FAIM2 is highly expressed in neural tissues .

• Expected results: Look for a primary band at approximately 30 kDa, which is the observed molecular weight for FAIM2 despite the calculated weight of 35 kDa .

Importantly, researchers should verify that their blocking and washing conditions don't interfere with antibody binding, especially when working with membrane-associated proteins like FAIM2.

What approaches are most effective for studying FAIM2 protein-protein interactions?

Based on published research, several methodological approaches have proven effective for studying FAIM2 protein-protein interactions:

• Co-immunoprecipitation (Co-IP): This has been successfully used to demonstrate FAIM2 interactions with p53 and HSP90 following FAS receptor activation . For Co-IP experiments:

  • Use mild lysis buffers (containing 1% NP-40 or 0.5% Triton X-100) to preserve protein complexes

  • Pre-clear lysates to reduce non-specific binding

  • Include appropriate negative controls (IgG or irrelevant antibody pulldowns)

• Proteomics analysis after immunoprecipitation: This unbiased approach has identified 31-71 potential FAIM2-interacting proteins in stressed photoreceptor cells . Studies have shown that:

  • FAS-ligand treatment enhances the interaction between FAIM2 and both p53 and HSP90

  • Phosphorylation of FAIM2 appears to be critical for its interaction with HSP90

• Proximity ligation assays: For detecting in situ protein interactions in fixed cells or tissues, this method can visualize FAIM2 interactions with FAS receptor components.

• FRET/BRET assays: For studying dynamic interactions in living cells, these techniques can reveal temporal aspects of FAIM2 associations with other proteins during apoptotic signaling.

Research has revealed that FAIM2 phosphorylation by JNK following FAS-ligand exposure increases its association with the FAS receptor and decreases its proteasome-mediated degradation . This phosphorylation-dependent interaction mechanism should be considered when designing experiments to study FAIM2 protein interactions.

How does FAIM2 expression correlate with cancer prognosis and immune infiltration?

Pan-cancer analysis has revealed important correlations between FAIM2 expression, prognosis, and immune infiltration that researchers should consider when designing cancer studies:

FAIM2 expression is generally downregulated in most tumor types compared to adjacent normal tissues . Interestingly, higher FAIM2 expression correlates with better prognosis in several cancer types, suggesting a potential tumor suppressor role . The relationship between FAIM2 expression and immune parameters varies by cancer type:

• Positive correlation with immune infiltration: In BLCA, HNSC, KIRP, LIHC, LUSC, PRAD, SKCM, THCA, and UVM, FAIM2 expression positively correlates with both ImmuneScore and StromalScore, suggesting FAIM2 may promote immune cell infiltration in these cancers .

• Negative correlation with immune infiltration: In LGG (Lower Grade Glioma), FAIM2 expression strongly negatively correlates with immune scores .

• CD8+ T cell infiltration: FAIM2 expression positively correlates with CD8+ T cell infiltration in most TCGA tumors, particularly in BRCA-Basal, KIRP, LUSC, PAAD, and SKCM based on CIBERSORT-ABS and MCPCOUNTER algorithms .

• Cancer-associated fibroblasts (CAFs): Positive correlations exist between FAIM2 expression and CAF presence in most tumors except LGG, GBM, and PCPG .

• Myeloid-derived suppressor cells (MDSCs): FAIM2 expression negatively correlates with MDSC presence in almost all tumor types via TIDE algorithms .

When designing studies to investigate FAIM2's role in cancer, researchers should account for these tissue-specific correlations and consider how FAIM2 might differentially affect various immune cell populations within the tumor microenvironment.

What methodological approaches should be used to investigate FAIM2's role in cancer metastasis?

Based on current research, particularly on FAIM2's role in non-small cell lung cancer (NSCLC) bone metastasis, researchers should consider these methodological approaches:

• Cell-based assays: Implement the following functional assays to comprehensively evaluate FAIM2's impact on metastatic potential:

  • Proliferation assays (MTT, BrdU incorporation)

  • Migration assays (wound healing, transwell)

  • Invasion assays (Matrigel-coated transwell)

  • Anoikis resistance assessment

  • Adhesion assays to relevant substrates (e.g., osteoblasts for bone metastasis studies)

• Molecular pathway analysis: Examine FAIM2's influence on:

  • Epithelial-mesenchymal transition (EMT) markers (E-cadherin, N-cadherin, Vimentin)

  • Wnt/β-catenin signaling pathway components

  • Expression of metastasis-related genes

• In vivo metastasis models: Establish appropriate animal models to validate in vitro findings:

  • Orthotopic xenograft models

  • Tail vein injection for experimental metastasis

  • Intracardiac injection for bone metastasis studies

• Patient sample analysis: Correlate FAIM2 expression in primary tumors with:

  • Metastatic status

  • Site-specific metastasis (e.g., bone vs. other sites)

  • Patient outcomes

For NSCLC specifically, research has utilized HARA cells with FAIM2 overexpression and HARA-B4 cells with FAIM2 knockdown to investigate effects on metastatic properties . Immunohistochemistry has been effectively employed to assess FAIM2 expression in normal lung tissue versus NSCLC tissue with or without bone metastasis .

When designing these experiments, researchers should include appropriate controls, such as vector-only transfections for overexpression studies and non-targeting siRNA/shRNA for knockdown experiments.

How should researchers design experiments to study FAIM2's neuroprotective functions?

FAIM2's neuroprotective role has been primarily studied in the context of FAS-mediated apoptosis in neural tissues, particularly photoreceptors. Based on published methodologies, researchers should consider these experimental approaches:

• Stress induction models:

  • Retinal detachment models for studying photoreceptor apoptosis

  • Oxygen-glucose deprivation for neuronal stress

  • FAS-ligand treatment to activate the extrinsic apoptotic pathway

• Genetic manipulation approaches:

  • Faim2 knockout mice have shown accelerated photoreceptor apoptosis following experimental retinal detachment

  • FAIM2 overexpression systems to assess neuroprotective effects

  • CRISPR-Cas9 gene editing for precise modification of FAIM2 sequences

• Apoptosis assessment:

  • TUNEL staining for quantifying apoptotic cells (knockout animals showed nearly doubled numbers of dying cells compared to wild-type at peak apoptotic activity)

  • Caspase activity assays

  • Annexin V/PI staining for flow cytometry

• Subcellular localization studies:

  • Immunofluorescence microscopy to track FAIM2 redistribution during stress

  • Subcellular fractionation followed by Western blotting

  • Live-cell imaging with fluorescently tagged FAIM2

• Phosphorylation analysis:

  • Phospho-specific antibodies or Phos-tag gels to detect JNK-mediated FAIM2 phosphorylation

  • Phosphomimetic and phospho-dead mutants to assess functional importance of phosphorylation

Research has shown that retinal detachment increases FAIM2 levels in photoreceptors, with higher amounts detected at the tips of outer segments . Activation of FAS receptor leads to JNK-mediated FAIM2 phosphorylation, decreased proteasome-mediated degradation, and increased association with the FAS receptor . These findings suggest that researchers should focus on both expression levels and post-translational modifications when studying FAIM2's neuroprotective functions.

What are the key considerations for analyzing FAIM2 protein interactions in neuronal stress models?

When analyzing FAIM2 protein interactions in neuronal stress models, researchers should consider several methodological factors:

• Timing of interaction analysis: FAIM2's neuroprotective effect appears to be transient , so time-course experiments are essential to capture dynamic interactions.

• Stress-dependent interactions: Research has shown that FAS-ligand treatment enhances FAIM2's interactions with specific proteins:

  • p53: Increased binding following FAS receptor activation

  • HSP90: Enhanced association of both total and phosphorylated HSP90 with FAIM2 during photoreceptor apoptosis

• Phosphorylation dependency: FAIM2/HSP90 interaction depends on the phosphorylation state of FAIM2 , suggesting researchers should:

  • Use phosphatase inhibitors in lysis buffers

  • Consider phosphomimetic mutants to stabilize interactions

  • Evaluate the effects of JNK inhibitors on protein interactions

• Technical approaches for interaction studies:

  • Co-immunoprecipitation with antibodies against FAIM2 or potential binding partners

  • Proximity ligation assay for in situ visualization of interactions in fixed tissues

  • Unbiased proteomics analysis following FAIM2 immunoprecipitation (previous studies identified 31-71 potential protein interactors)

• Downstream gene expression effects: Lack of FAIM2 leads to increased expression of pro-death genes like Fas and Ripk1 in the retina under physiologic conditions , suggesting researchers should complement protein interaction studies with gene expression analysis.

When designing these experiments, researchers should be mindful that FAIM2 interactions may differ between acute and chronic stress conditions, and that the cellular context (photoreceptors vs. other neuronal types) may influence the interaction profile.

How can researchers overcome challenges in detecting post-translational modifications of FAIM2?

Detecting post-translational modifications (PTMs) of FAIM2, particularly phosphorylation events that regulate its function, requires specialized approaches:

• Phosphorylation detection strategies:

  • Phos-tag SDS-PAGE: This technique can separate phosphorylated from non-phosphorylated FAIM2 based on mobility shifts

  • Phospho-specific antibodies: When available, these provide direct detection of specific phosphorylation sites

  • Mass spectrometry: For unbiased identification of phosphorylation sites and other PTMs

  • 2D gel electrophoresis: Can separate FAIM2 isoforms based on charge differences from phosphorylation

• Preserving phosphorylation during sample preparation:

  • Include phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate) in all lysis buffers

  • Maintain samples at 4°C during processing

  • Consider crosslinking approaches for transient modifications

• Experimental induction of phosphorylation:

  • JNK activation has been shown to induce FAIM2 phosphorylation

  • FAS-ligand treatment can trigger the pathway leading to FAIM2 phosphorylation

  • Anisomycin or UV treatment for JNK pathway activation

• Functional validation of phosphorylation:

  • Site-directed mutagenesis to create phosphomimetic (S→D or S→E) or phospho-dead (S→A) mutants

  • Compare proteasomal degradation rates between wild-type and mutant FAIM2

  • Assess interaction with FAS receptor and other binding partners using mutants

Research has demonstrated that JNK-mediated phosphorylation of FAIM2 following FAS-ligand exposure decreases its proteasome-mediated degradation and increases its association with the FAS receptor . Additionally, FAIM2 phosphorylation is required for interaction with HSP90 during stress conditions , highlighting the importance of properly detecting and characterizing these modifications.

What troubleshooting approaches are recommended when FAIM2 antibodies show inconsistent results?

When facing inconsistent results with FAIM2 antibodies, researchers should systematically troubleshoot using these approaches:

• Antibody validation:

  • Confirm antibody specificity using positive controls (brain tissue for FAIM2)

  • Test antibody performance in FAIM2 knockout/knockdown samples

  • Consider using multiple antibodies targeting different epitopes of FAIM2

• Sample preparation optimization:

  • FAIM2 is a membrane-associated protein found in the Golgi, ER, and plasma membrane , so membrane protein extraction protocols may be necessary

  • Test different lysis buffers (RIPA vs. NP-40 vs. Triton X-100)

  • Optimize protein denaturation conditions (temperature, reducing agents)

• Protocol adjustments for Western blotting:

  • Titrate antibody concentration (recommended range: 1:500-1:3000)

  • Modify blocking conditions (5% milk vs. 3-5% BSA)

  • Adjust incubation times and temperatures

  • Test different transfer methods for membrane proteins

• Consider protein characteristics:

  • Note the discrepancy between calculated (35 kDa) and observed (30 kDa) molecular weights

  • Be aware of potential post-translational modifications affecting epitope recognition

  • FAIM2 expression is tissue-specific, with highest levels in nervous system

• Alternative detection approaches:

  • If Western blotting is problematic, consider immunoprecipitation followed by mass spectrometry

  • For tissue localization, try RNAscope or in situ hybridization as alternatives to IHC

  • Use tagged recombinant FAIM2 constructs when possible

Researchers should note that FAIM2 expression can be stress-responsive, with levels increasing during conditions like retinal detachment . Therefore, experimental conditions that affect cellular stress levels may impact FAIM2 detection. Additionally, since FAIM2 is subject to proteasomal degradation, which is reduced by phosphorylation , treatment with proteasome inhibitors might enhance detection in some experimental contexts.

How might researchers design experiments to explore FAIM2's potential as a therapeutic target?

Based on current understanding of FAIM2's roles in neuroprotection and cancer, researchers might design experiments to explore its therapeutic potential using these methodological approaches:

• For neurological disease applications:

  • FAIM2 stabilization strategies: Design peptides or small molecules that enhance FAIM2 phosphorylation or inhibit its proteasomal degradation

  • Targeted delivery systems: Develop methods to increase FAIM2 expression specifically in neurons at risk

  • JNK pathway modulators: Test compounds that enhance JNK-mediated FAIM2 phosphorylation without triggering other pro-apoptotic JNK functions

  • Efficacy assessment: Measure neuroprotection in models of retinal detachment, cerebral ischemia, or neurodegenerative diseases

• For cancer applications:

  • Context-dependent targeting: Since FAIM2 appears to have different roles across cancer types, develop tissue-specific approaches

  • Immune modulation: Design experiments to determine if enhancing FAIM2 expression can increase CD8+ T cell infiltration in tumors

  • Combinatorial approaches: Test FAIM2-targeting strategies alongside immune checkpoint inhibitors

  • Biomarker development: Validate FAIM2 expression as a prognostic biomarker across cancer types

• Experimental validation approaches:

  • Use genetic models (conditional FAIM2 knockout or overexpression)

  • Develop inducible systems for temporal control of FAIM2 expression

  • Implement tissue-specific targeting to avoid unwanted effects in the nervous system when targeting FAIM2 in cancer

Research indicates that FAIM2 protects uniquely from cell death induced by Fas but not by TNFα , suggesting therapeutic approaches should focus on contexts where FAS-mediated apoptosis is the primary mechanism of cell death or immune evasion.

What methodological considerations are important when studying species-specific differences in FAIM2 function?

When investigating species-specific differences in FAIM2 function, researchers should consider these methodological approaches:

• Cross-species sequence and structural analysis:

  • FAIM2 is evolutionarily conserved , but species-specific variations may affect antibody recognition and protein interactions

  • Conduct sequence alignments across species of interest

  • Identify conserved functional domains and species-specific variations

• Antibody selection and validation:

  • The FAIM2 antibody (15211-1-AP) shows reactivity with human, mouse, and rat samples

  • For studies in other species, validate antibody cross-reactivity

  • Consider developing species-specific antibodies for non-reactive species

• Model system selection:

  • Mouse models have been effective for studying FAIM2 in retinal detachment

  • For neurological studies, consider species differences in nervous system architecture

  • In cancer research, be aware that tumor microenvironments may vary significantly across species

• Functional conservation testing:

  • Complementation studies with FAIM2 from different species

  • Compare phosphorylation patterns and protein interactions across species

  • Assess whether regulatory mechanisms (JNK-mediated phosphorylation, proteasomal degradation) are conserved

• Experimental controls for cross-species studies:

  • Include species-matched positive controls

  • Use species-specific reference genes/proteins for normalization

  • Consider codon optimization for cross-species expression experiments