FLVCR2 Antibody

What is FLVCR2 and what are its primary physiological functions?

FLVCR2 is a transmembrane protein that functions as a multispecific transporter with several identified roles:

• Choline uniporter that specifically mediates choline uptake at the blood-brain barrier, responsible for the majority of choline transport from circulation into the brain

• Ethanolamine transporter across the plasma membrane

• Heme importer, with binding capabilities that can be competed by free hemin

• Previously identified as a receptor for FY981 feline leukemia virus

FLVCR2 belongs to the major facilitator superfamily (MFS) of transporters, with an architecture consisting of 12 transmembrane domains. Recent cryo-EM structural studies have revealed its conformational dynamics during substrate transport, showing both inward-facing and outward-facing conformations .

What applications are FLVCR2 antibodies commonly validated for?

Commercial FLVCR2 antibodies have been validated for multiple applications:

Most commercially available antibodies show reactivity against human FLVCR2, with some cross-reactivity to mouse, dog, and other mammalian species .

What are key considerations for selecting an appropriate FLVCR2 antibody?

When selecting an FLVCR2 antibody, researchers should consider:

• Epitope specificity: Different antibodies target distinct regions of FLVCR2 (N-terminal, C-terminal, or specific internal domains). For example, some antibodies target amino acids 1-100, while others target the C-terminal region (amino acids 478-507) . The epitope location may affect detection depending on protein conformation or processing.

• Species reactivity: Verify cross-reactivity with your experimental species. Most FLVCR2 antibodies are validated for human samples, with variable cross-reactivity to mouse, dog, horse, and other mammals .

• Application compatibility: Ensure the antibody is validated for your specific application. Some antibodies work well for Western blotting but may not be suitable for immunohistochemistry or flow cytometry .

• Clonality: Polyclonal antibodies may provide higher sensitivity but potentially lower specificity compared to monoclonal antibodies. Most commercial FLVCR2 antibodies are polyclonal, typically produced in rabbits .

• Validation data: Review available validation data, including Western blot images, immunohistochemistry results, and any available knockout/knockdown controls .

How can I optimize FLVCR2 detection by Western blotting?

For optimal detection of FLVCR2 by Western blotting:

• Sample preparation:

  • Use fresh tissue/cells and include protease inhibitors during lysis

  • Avoid excessive heating of samples as FLVCR2 is a transmembrane protein

  • Consider membrane protein extraction methods for enrichment

• Recommended antibody concentrations:

  • Typical working dilutions range from 0.04-0.4 μg/mL or 1:1000-1:5000 dilution

• Blocking conditions:

  • 5% non-fat milk or BSA in TBST is generally effective

  • For phospho-specific detection, BSA is preferred over milk

• Troubleshooting multiple bands:

  • FLVCR2 may show post-translational modifications or processing

  • Verify expected molecular weight (~55-60 kDa)

  • Consider using reducing/non-reducing conditions to optimize detection

• Controls:

  • Include positive control tissues known to express FLVCR2 (brain endothelial cells, placenta)

  • Consider siRNA knockdown controls to verify specificity

What is the tissue distribution of FLVCR2 expression?

FLVCR2 expression has been documented in:

• Brain: Particularly in brain endothelial cells forming the blood-brain barrier

• Placenta: High expression observed

• Liver: Moderate expression

• Kidney: Detectable expression

Expression analysis can be performed using quantitative PCR with primers targeting FLVCR2 transcripts, as described in previous studies (e.g., primers 5′-TTGTCCTGGTGTTTAGCTGCTACT-3′ and 5′-AGTCAATGGCAAAGGCACTGACAC-3′) .

mRNA expression should be normalized against housekeeping genes such as actin (primers ActB-F [5′-TGCGTGACATTAAGGAGAAG-3′] and ActB-R [5′-AGGAAGGAAGGCTGGAAGAG-3′]) .

How can I design experiments to investigate FLVCR2's dual role in heme and choline transport?

To differentiate between FLVCR2's roles in heme versus choline transport:

• Transport assays using radioactive or fluorescent substrates:

  • For heme transport: Use radiolabeled hemin or zinc mesoporphyrin (ZnMP) as a fluorescent heme analog

  • For choline transport: Use [³H]choline chloride in uptake assays

  • For ethanolamine transport: Use radiolabeled ethanolamine

• Competition studies:

  • Test whether excess unlabeled choline competes with heme uptake and vice versa

  • Include known inhibitors of each pathway as controls

• Site-directed mutagenesis:

  • Based on structural data, mutate residues in the substrate-binding pocket predicted to affect one transport function but not the other

  • Evaluate transport activity for both substrates after mutation

• Cellular models:

  • Compare transport in cell types with differing physiological demands for heme versus choline

  • Consider brain endothelial cells for choline transport and erythroid precursors for heme transport studies

• Concentration-dependent kinetics:

  • Determine Km and Vmax for each substrate to assess transport preferences

  • Evaluate pH dependence, as choline transport is enhanced by inwardly directed proton gradients

What experimental approaches are recommended for studying FLVCR2 in the context of Fowler syndrome?

Fowler syndrome is a proliferative vascular disorder of the brain associated with FLVCR2 mutations . To study FLVCR2 in this context:

• Mouse models:

  • Conditional knockout models using endothelial-specific drivers (e.g., Cdh5CreER)

  • Analyze vascular phenotypes, focusing on brain angiogenesis and the blood-brain barrier integrity

  • Time-dependent analysis during critical developmental windows

• Human patient mutations:

  • Introduce Fowler syndrome-associated FLVCR2 mutations into cellular models using CRISPR/Cas9

  • Assess transport activity, protein localization, and protein stability

• Vascular sprouting assays:

  • Brain endothelial cell spheroid sprouting assays

  • Retinal angiogenesis models

  • Evaluate tip cell formation and sprouting behavior

• Histopathological analysis:

  • Compare glomeruloid vasculopathy in patient samples versus animal models

  • Investigate hypoxia markers in affected tissues

• Mechanistic studies:

  • Investigate downstream signaling pathways affected by FLVCR2 dysfunction

  • Analyze the expression of angiogenic factors in FLVCR2-deficient endothelial cells

How can I distinguish between FLVCR1 and FLVCR2 functions in my experimental model?

FLVCR1 and FLVCR2 are related proteins with distinct transport directions and somewhat overlapping substrates . To distinguish their functions:

• Selective genetic manipulation:

  • Use specific siRNAs for knockdown (e.g., FLVCR2-specific siRNAs: S1 (FLVCR2HSS124723) and S2 (FLVCR2HSS124724))

  • Employ CRISPR/Cas9 for selective knockout

  • Create rescue experiments with wild-type versus mutant constructs

• Transport directionality:

  • FLVCR1 primarily functions as an exporter

  • FLVCR2 functions as an importer

  • Design experiments to measure influx versus efflux of substrates

• Differential substrate affinity:

  • Compare transport kinetics for heme, choline, and ethanolamine

  • Assess competitive inhibition patterns

• Structural differences:

  • Utilize the recently resolved structures of both transporters to design selective inhibitors or binding studies

  • Target regions with low sequence homology between the two proteins

• Expression pattern analysis:

  • Compare tissue-specific expression profiles

  • Determine subcellular localization differences

What are optimal methods for FLVCR2 knockdown/knockout studies?

For effective manipulation of FLVCR2 expression:

• siRNA knockdown:

  • Validated FLVCR2-specific siRNAs include sequences S1 (FLVCR2HSS124723) and S2 (FLVCR2HSS124724)

  • Optimal transfection conditions depend on cell type (typically 20-50 nM siRNA)

  • Validate knockdown by qPCR and Western blot at 48-72 hours post-transfection

• CRISPR/Cas9 knockout:

  • Target early exons to ensure complete loss of function

  • Use multiple guide RNAs to increase efficiency

  • Validate knockout by sequencing, Western blot, and functional assays

• Conditional approaches:

  • For in vivo studies, use conditional knockout systems (e.g., Flvcr2fl/fl;Cdh5CreER mouse model)

  • Induce knockout with tamoxifen at specific developmental stages

  • For cellular models, consider inducible CRISPR or shRNA systems

• Phenotypic validation:

  • Measure transport of relevant substrates (heme, choline, ethanolamine)

  • Assess cell viability, as complete knockout may be lethal in some cell types

  • For brain endothelial cells, evaluate angiogenic capacity and barrier formation

• Controls and rescue experiments:

  • Include scrambled siRNA controls

  • Perform rescue experiments with wild-type or mutant FLVCR2 expression constructs

  • Consider species-specific approaches (human FLVCR2 may not fully rescue mouse Flvcr2 knockout)

How can I assess FLVCR2 structure-function relationships in transport studies?

Recent structural data provides opportunities to investigate structure-function relationships :

• Site-directed mutagenesis based on structural insights:

  • Target residues in the substrate-binding pocket identified by cryo-EM structures

  • Modify residues involved in conformational changes between inward-facing and outward-facing states

  • Mutate interface residues between N and C domains that may affect transport dynamics

• Construct design:

  • Create domain-swapping chimeras between FLVCR1 and FLVCR2 to identify determinants of transport direction

  • Generate truncation mutants to assess the role of cytoplasmic domains

  • Introduce tags at non-conserved regions to minimize functional disruption

• Transport assays:

  • Compare wild-type versus mutant constructs in radioligand uptake assays

  • Assess conformational dynamics using accessibility studies (e.g., cysteine scanning)

  • Measure binding affinities using competition assays with labeled substrates

• Molecular dynamics simulations:

  • Utilize the solved structures for in silico modeling of substrate binding and transport

  • Predict effects of mutations before experimental validation

  • Model transition states between conformations

• Inhibitor studies:

  • Design small molecules targeting specific structural features

  • Test competitive versus non-competitive inhibition to probe binding sites

  • Use FY981 FeLV envelope protein as a specific inhibitor of FLVCR2

What methods are recommended for validating the specificity of FLVCR2 antibodies?

Rigorous validation is essential for antibody-based studies:

• Genetic approaches:

  • Test antibody reactivity in FLVCR2 knockout or knockdown samples

  • Use siRNA-treated cells as negative controls in Western blot and immunostaining

  • Overexpress FLVCR2 as a positive control

• Peptide competition:

  • Pre-incubate antibody with the immunizing peptide before application

  • Compare signal with and without peptide competition

  • Use related but distinct peptides as specificity controls

• Cross-reactivity assessment:

  • Test reactivity against related proteins (especially FLVCR1)

  • Evaluate species cross-reactivity if working with non-human models

  • Consider testing in tissues known to express or lack FLVCR2

• Multiple antibody approach:

  • Use antibodies targeting different epitopes (e.g., N-terminal versus C-terminal)

  • Compare staining patterns and signal intensities

  • Confirm that different antibodies yield consistent results

• Application-specific validation:

  • For Western blotting: Verify band size, test reduced/non-reduced conditions

  • For IHC/IF: Include appropriate negative controls, test antigen retrieval methods

  • For flow cytometry: Compare surface versus permeabilized staining

How should I design experiments to study FLVCR2's role in brain angiogenesis?

FLVCR2 has been implicated in brain vascular development, with knockout leading to defects in angiogenic sprouting :

• In vivo models:

  • Conditional endothelial-specific knockout (Flvcr2fl/fl;Cdh5CreER)

  • Developmental timing analysis focusing on critical windows of brain vascularization

  • Retinal angiogenesis as an accessible model of CNS vascular development

• Ex vivo approaches:

  • Brain slice cultures from control versus FLVCR2-deficient animals

  • Brain organoid models with genetically modified endothelial cells

  • Aortic ring sprouting assays to assess angiogenic potential

• In vitro endothelial studies:

  • Spheroid sprouting assays using brain-derived endothelial cells

  • Tube formation assays on Matrigel

  • Transwell migration and proliferation assays

• Molecular readouts:

  • Tip cell marker expression (e.g., DLL4, VEGFR2)

  • Angiogenic factor expression profiling

  • Analysis of vessel morphology (branching, diameter, coverage)

• Functional consequences:

  • Tissue hypoxia assessment using pimonidazole staining or HIF-1α immunostaining

  • Evaluation of blood-brain barrier integrity using tracer molecules

  • Assessment of neurological consequences and hydrocephalus development

What are the most reliable detection methods for monitoring FLVCR2-mediated transport?

To quantify FLVCR2 transport activity:

• Radioactive substrate uptake:

  • [³H]choline chloride for choline transport

  • ⁵⁵Fe-labeled hemin for heme transport

  • Set up time-course experiments to establish linear uptake ranges

• Fluorescent substrate analogs:

  • Zinc mesoporphyrin (ZnMP) as a fluorescent heme analog

  • NBD-labeled choline derivatives for microscopy-based uptake

  • Flow cytometry for quantitative single-cell analysis

• Electrophysiological measurements:

  • Xenopus oocyte expression system for two-electrode voltage clamp recordings

  • Patch-clamp techniques for measuring substrate-induced currents

  • Membrane potential-sensitive dyes for high-throughput screening

• Transport inhibition controls:

  • FY981 FeLV envelope protein as a specific inhibitor of FLVCR2

  • Structural analogs as competitive inhibitors

  • Modulation by pH gradients (proton-enhanced transport)

• Data analysis considerations:

  • Normalize for protein expression levels

  • Calculate kinetic parameters (Km, Vmax)

  • Account for endogenous transport by using proper controls

How can apparent contradictions in FLVCR2 function be reconciled experimentally?

The literature contains some apparent discrepancies regarding FLVCR2 function:

• Heme transport versus choline/ethanolamine transport:

  • Design comparative substrate preference studies using the same experimental system

  • Determine whether transport functions are mutually exclusive or can occur simultaneously

  • Investigate whether substrate preference depends on cell type or physiological conditions

• Role in blood-brain barrier function versus development:

  • Recent findings show that FLVCR2 is necessary for brain angiogenesis but surprisingly dispensable for blood-brain barrier maintenance

  • Design developmental time-course experiments to distinguish between roles in different processes

  • Use tissue-specific and temporally controlled knockout models

• Reconciling transport mechanisms:

  • Investigate whether heme and choline binding sites overlap or are distinct

  • Determine if conformational changes induced by one substrate affect binding of others

  • Use the recently determined structures to inform experimental design

• Pathological mechanisms in Fowler syndrome:

  • Compare effects of complete loss versus partial dysfunction of transport activity

  • Investigate whether specific mutations differentially affect heme versus choline transport

  • Determine which transport function is most critical for brain vascular development

What are the optimal conditions for immunoprecipitation of FLVCR2?

For successful immunoprecipitation of FLVCR2:

• Lysis buffer optimization:

  • Use detergent-based buffers suitable for membrane proteins (e.g., 1% NP-40, CHAPS, or digitonin)

  • Include protease inhibitors to prevent degradation

  • Consider using cross-linking agents to stabilize protein complexes

• Antibody selection:

  • Choose antibodies validated for immunoprecipitation

  • Consider epitope accessibility in the native protein

  • Use tagged constructs (e.g., HA-tagged FLVCR2) if native antibodies are insufficient

• Protocol considerations:

  • Pre-clear lysates to reduce non-specific binding

  • Optimize antibody concentration and incubation time

  • For weak interactions, consider formaldehyde or DSP cross-linking before lysis

• Controls:

  • Include isotype control antibodies

  • Use lysates from FLVCR2-knockout cells as negative controls

  • Verify pulldown efficiency by comparing input, unbound, and eluted fractions

• Detection methods:

  • Western blotting using a different FLVCR2 antibody than used for IP

  • Mass spectrometry for identifying interaction partners

  • Activity assays using precipitated protein (e.g., heme binding)

How can I design experiments to investigate FLVCR2 in multiple species models?

For comparative studies across species:

• Sequence homology analysis:

  • Human FLVCR2 shows variable conservation across species (Dog: 93%, Horse: 86%, Pig: 93%, Rabbit: 79%)

  • Align sequences to identify conserved domains for targeting

  • Consider species-specific differences in epitopes when selecting antibodies

• Cross-species antibody validation:

  • Test reactivity against recombinant proteins from each species

  • Validate in tissues from different species using identical protocols

  • Consider generating species-specific antibodies if cross-reactivity is poor

• Functional conservation assessment:

  • Compare transport kinetics in cells from different species

  • Use cross-species complementation studies (e.g., human FLVCR2 in mouse knockout cells)

  • Investigate species-specific interaction partners

• Model system selection:

  • Mouse models for in vivo developmental studies

  • Xenopus oocytes for controlled transport studies

  • Human cell lines for disease-relevant mechanisms

• Species-specific considerations:

  • Account for differences in tissue expression patterns

  • Consider variations in developmental timing

  • Be aware of potential differences in regulatory mechanisms

How should FLVCR2 antibody validation data be critically evaluated?

When assessing antibody validation data:

• Western blot evaluation:

  • Verify single band at the expected molecular weight (~55-60 kDa)

  • Check for additional bands that might indicate cross-reactivity

  • Examine knockout/knockdown controls showing band disappearance

• Immunostaining pattern assessment:

  • Confirm localization consistent with membrane protein (plasma membrane, possibly some ER)

  • Compare with known expression patterns in tissues (e.g., brain endothelial cells)

  • Evaluate background staining and signal-to-noise ratio

• Quantitative considerations:

  • Look for dose-response relationships in antibody concentration experiments

  • Assess reproducibility across multiple experiments

  • Compare results with antibodies targeting different epitopes

• Red flags in validation data:

  • Inconsistent patterns between applications (e.g., multiple bands in WB but clean IHC)

  • Lack of appropriate negative controls

  • Significant lot-to-lot variability

  • Discrepancies with published literature

• Independent validation approaches:

  • Correlation with mRNA expression data

  • Confirmation using orthogonal methods (e.g., mass spectrometry)

  • Functional validation (e.g., transport activity correlating with staining intensity)

What are common pitfalls in FLVCR2 expression and functional studies?

Researchers should be aware of these common challenges:

• Expression challenges:

  • Overexpression toxicity due to altered membrane composition or transport activity

  • Improper folding or trafficking of overexpressed protein

  • Expression screening revealed variability among FLVCR2 variants

• Transport assay limitations:

  • Background transport by endogenous transporters

  • Substrate degradation during long incubations

  • Non-specific binding of hydrophobic substrates like heme

  • Toxicity of high heme concentrations

• Knockout/knockdown issues:

  • Developmental lethality of complete knockout

  • Compensatory upregulation of related transporters

  • Incomplete knockdown affecting interpretation

  • Secondary effects on cell viability or differentiation

• Antibody-related problems:

  • Non-specific binding

  • Epitope masking in certain applications

  • Batch-to-batch variability

  • Limited cross-reactivity with non-human species

• Interpretation challenges:

  • Distinguishing primary from secondary effects

  • Reconciling different functions (heme vs. choline transport)

  • Extrapolating from in vitro to in vivo significance

  • Connecting transport defects to vascular phenotypes

How can biochemical assays be optimized to study FLVCR2 protein-protein interactions?

To investigate FLVCR2 interactions with other proteins:

• Pulldown assays:

  • Use hemin-agarose to pull down FLVCR2 and identify co-purifying proteins

  • Include competition with free substrate to verify specificity

  • Consider mild detergents to preserve membrane protein complexes

• Co-immunoprecipitation approaches:

  • Use tagged FLVCR2 constructs if native antibodies are limiting

  • Cross-link before lysis to capture transient interactions

  • Validate by reciprocal IP where possible

• Proximity labeling techniques:

  • BioID or TurboID fusion proteins to identify proximal proteins

  • APEX2 for temporally controlled labeling

  • Targeted to specific cellular compartments to reduce background

• FRET/BRET approaches:

  • Tag FLVCR2 and potential interaction partners with compatible fluorophores

  • Measure energy transfer as indication of proximity

  • Use for live-cell interaction dynamics

• Control experiments:

  • Include substrate-free conditions

  • Test structurally similar non-substrate molecules

  • Use FLVCR2 mutants defective in transport but properly localized

How can I reconcile contradictory results between different FLVCR2 antibodies?

When different antibodies yield inconsistent results:

• Epitope mapping considerations:

  • Compare the specific regions targeted by each antibody

  • Consider whether certain epitopes may be masked in particular applications

  • Evaluate whether post-translational modifications might affect epitope recognition

• Technical validation:

  • Test all antibodies under identical conditions

  • Perform peptide competition for each antibody

  • Validate in knockout/knockdown systems

• Application-specific differences:

  • Some antibodies work well for denatured proteins (WB) but poorly for native forms (IP)

  • Fixation methods in IHC/IF may differently affect epitope accessibility

  • Consider native versus reducing conditions for Western blotting

• Resolution strategies:

  • Use multiple antibodies targeting different regions and look for consistent results

  • Generate new reagents with improved specificity

  • Employ orthogonal detection methods (e.g., mass spectrometry)

  • Use tagged constructs when studying overexpression systems

• Documentation and reporting:

  • Clearly document all antibody details (source, catalog number, lot)

  • Note specific conditions that affect antibody performance

  • Report inconsistencies rather than selecting only "working" antibodies

What emerging technologies could advance FLVCR2 research?

Cutting-edge approaches for FLVCR2 investigation include:

• Structural biology advances:

  • Recent cryo-EM structures provide templates for detailed mechanistic studies

  • Time-resolved structural methods to capture transport dynamics

  • Computational modeling of substrate binding and translocation

• Single-cell technologies:

  • Single-cell RNA-seq to identify cell populations expressing FLVCR2

  • Spatial transcriptomics to map expression in complex tissues like brain

  • CyTOF for protein-level analysis in heterogeneous populations

• Advanced genome editing:

  • Base editing or prime editing for introducing precise mutations

  • Inducible, cell-type-specific CRISPR systems

  • Knock-in reporters to monitor FLVCR2 expression and localization

• In vitro models:

  • Brain organoids with vascular components

  • Organ-on-chip devices to study blood-brain barrier function

  • Patient-derived iPSCs differentiated to relevant cell types

• Therapeutic approaches:

  • Structure-based drug design targeting FLVCR2

  • Gene therapy approaches for Fowler syndrome

  • Small molecule modulators of specific transport functions

What is the significance of recent structural insights into FLVCR2 function?

Recent structural studies have revealed:

• Conformational dynamics:

  • FLVCR2 adopts both inward-facing and outward-facing conformations

  • Structural basis for the alternating access mechanism

  • Domain movements during the transport cycle

• Substrate specificity determinants:

  • Molecular architecture of the substrate-binding site

  • Structural basis for recognizing different substrates (choline, ethanolamine)

  • Comparison with related transporters (FLVCR1)

• Disease-relevant insights:

  • Structural context of mutations associated with Fowler syndrome

  • Mechanism of substrate translocation

  • Potential for structure-based drug design

• Experimental opportunities:

  • Rational design of mutants to probe specific aspects of transport

  • Development of conformation-specific antibodies

  • Structure-guided design of high-affinity inhibitors

• Integration with functional data:

  • Connection between structural features and transport kinetics

  • Explanation for dual substrate specificity

  • Insights into the evolution of transport function