CAT1 Antibody

What is CAT1 and what cellular functions does it perform?

CAT1 (SLC7A1) is a transmembrane protein with 14 putative transmembrane domains that functions as a transporter responsible for the uptake of cationic amino acids (arginine, lysine, and ornithine) essential for cellular growth . As a member of the solute carrier family, CAT1 plays a critical role in amino acid metabolism and cellular nutrition. The protein is encoded by the SLC7A1 gene, which is located on chromosome 13q12.3, a region that has been identified through comparative genomic hybridization (CGH) as having a high frequency of gene amplifications in colorectal cancers . CAT1 is expressed in a wide variety of cell types across multiple species, suggesting its fundamental importance in cellular physiology.

How should researchers validate CAT1 antibodies for experimental use?

For proper validation of CAT1 antibodies, researchers should employ multiple approaches:

• Western Blot Validation: Compare expression in CAT1-transfected versus non-transfected cells. For example, analysis can be performed using SLC7A1-transfected 293T cell lysate (expected molecular weight: 67.6 KDa) compared against non-transfected lysate as a negative control .

• Knockdown Controls: Utilize siRNA targeting CAT1/SLC7A1 and evaluate antibody specificity by assessing the reduction in signal. This approach has been successfully implemented in studies where CAT1 expression was knocked down in CC81-GREMG cells using Silencer select siRNAs targeting the CAT1/SLC7A1 gene .

• Cross-reactivity Assessment: Test antibody reactivity across species if performing comparative studies. Evidence shows CAT1 antibodies can detect the protein across multiple species including human, feline, bovine, and porcine cell lines .

• Immunohistochemical Validation: When performing IHC, use isotype-matched controls (e.g., rat IgG (γ2b/κ)) and establish scoring systems similar to standard procedures like the HER2 test, with scores ranging from 0-3 indicating negative, weak, intermediate, or strong CAT1 expression .

What expression patterns of CAT1 have been observed across different cell types?

CAT1 expression varies significantly across cell lines and tissue types, with notable patterns:

Western blot analysis with anti-CAT1 antibodies has demonstrated expression in multiple cell lines across six animal species, with CHO-K1 cells being the notable exception with undetectable CAT1 protein levels . This widespread expression pattern correlates with CAT1's fundamental role in amino acid transport and highlights its conservation across species.

How do CAT1 expression levels correlate with disease states?

CAT1 expression shows significant alterations in multiple disease states:

In colorectal cancer (CRC), CAT1 mRNA is overexpressed in more than 70% of human samples compared to adjacent normal tissues . This overexpression correlates with the high frequency of gene amplifications at the chromosomal region 13q12.3 where SLC7A1 is located . The differential expression between tumor and normal tissues makes CAT1 a potential diagnostic marker and therapeutic target.

In rheumatoid arthritis (RA), CAT1 promotes abnormal proliferation of fibroblast-like synoviocytes (FLSs) in the synovial lining layer, which is a primary cause of synovial hyperplasia and joint destruction . This suggests a link between amino acid metabolism abnormalities and FLS proliferation in RA pathogenesis.

Metabolic studies have also identified CAT1 expression changes in cardiac hypertrophy following transverse aortic constriction (TAC), with western immunoblotting showing altered expression alongside other network molecules (HDGF, BCL2) and regulatory miRNAs (miRNA214 and miRNA378) .

What mechanisms underlie CAT1's role in cancer progression?

CAT1's involvement in cancer progression, particularly colorectal cancer (CRC), operates through multiple mechanisms:

• Oncogene Addiction: RNA interference-mediated knockdown of CAT1 inhibits the cell growth of CRCs, suggesting that cancer cells become dependent on CAT1 function for survival and proliferation .

• Amino Acid Metabolism: As CAT1 transports cationic amino acids essential for cellular growth (arginine, lysine, and ornithine), its overexpression potentially provides cancer cells with increased access to these building blocks for protein synthesis and other metabolic functions, supporting their rapid proliferation .

• Cell Migration: Anti-CAT1 monoclonal antibodies demonstrate migration inhibition activity against CRC cell lines in modified Boyden chamber assays. When LS-LM4 cells are treated with anti-CAT1 mAb (CA2) before seeding, their migration toward vitronectin (VN) and neuregulin 1 (NRG-1) is significantly reduced . This suggests that CAT1 plays a role in cancer cell motility, which is crucial for metastasis.

• Signal Transduction: Though the exact signaling pathways remain to be fully elucidated, the interaction between CAT1 and extracellular matrix components (like vitronectin) suggests its involvement in integrin-mediated signaling, which regulates cellular adhesion, migration, and invasion—key processes in cancer progression .

Research using comparative genomic hybridization has identified SLC7A1 in a chromosome region (13q12.3) with high frequency of gene amplifications, further supporting its role as a potential oncogene in CRC development .

How can researchers effectively use anti-CAT1 monoclonal antibodies for functional studies?

For effective functional studies using anti-CAT1 monoclonal antibodies (mAbs), researchers should consider these methodological approaches:

• Internalization Assays: To study antibody internalization, researchers can:

  • Perform microscopic studies by reacting CAT1-GFP-overexpressing cells (e.g., HEK293F) with anti-CAT1 mAbs (10 μg/mL) at 37°C for 1 hour

  • Fix cells with 4% PFA and stain with DAPI (0.5 μg/mL)

  • Acquire confocal fluorescence images to visualize internalization

  • For quantitative assessment, use flow cytometry after incubating cells with anti-CAT1 mAbs at 37°C versus 4°C (control) followed by PE-conjugated secondary antibody staining

• Migration Inhibition Assays: To assess effects on cell migration:

  • Use modified Boyden chambers with 8-μm pores

  • Fill lower chambers with medium containing 0.1% BSA, vitronectin (10 μg/mL), and neuregulin 1 (10 ng/mL)

  • Seed cells (e.g., 2 × 10^5 LS-LM4 cells) in upper chambers after pretreatment with anti-CAT1 mAb or control antibody

  • After incubation (20 hours), fix, stain, and count migrated cells on the lower surfaces of the filter

  • Compare migration rates between antibody-treated and control groups

• Antibody-Dependent Cellular Cytotoxicity (ADCC) Assays: Anti-CAT1 mAbs have demonstrated ADCC activity against CRC cell lines, suggesting their potential as therapeutic agents . Researchers can implement standardized ADCC assays to evaluate this function.

• In vivo Tumor Growth Inhibition Studies: For translational research:

  • Establish xenograft models using human CRC cell lines (e.g., HT29, SW-C4) in nude mice

  • Administer anti-CAT1 mAbs (e.g., CA2) and monitor tumor growth compared to controls

  • This approach has demonstrated that anti-CAT1 mAbs can inhibit the in vivo growth of human CRC tumors

What is the mechanism by which CAT1 functions as a viral receptor for BLV?

CAT1 functions as a cellular receptor for bovine leukemia virus (BLV) through specific molecular interactions and cellular processes:

• Direct Binding Interaction: CAT1 specifically binds to BLV particles on the cell surface through interaction with the viral envelope glycoprotein (Env). This binding is the initial step in the viral entry process .

• Colocalization: After binding, CAT1 colocalizes with the BLV Env in endomembrane compartments and at the membrane, suggesting involvement in the internalization and trafficking of viral particles .

• Cell Susceptibility Correlation: Cells expressing undetectable CAT1 levels (such as CHO-K1) are resistant to BLV infection but become highly susceptible upon CAT1 overexpression through transfection with a bCAT1/SLC7A1-expression plasmid .

• Knockdown Effects: When CAT1 is knocked down in permissive cells using siRNA, both binding to BLV particles and subsequent BLV infection are significantly reduced, confirming CAT1's essential role in viral entry .

• Cross-Species Functionality: CAT1 from various species shows no species specificity for BLV infection, which explains BLV's broad host range in vitro. This finding is particularly relevant for understanding the potential for cross-species transmission .

• Syncytium Formation: CAT1 mediates cell fusion leading to syncytium formation, which is a typical cytopathic effect observed in BLV infection and serves as a monitoring marker for infection in culture .

These mechanistic insights provide important information for developing strategies to prevent BLV spread and potential therapeutic interventions.

How can researchers distinguish between CAT1-mediated effects and other transport mechanisms in functional studies?

To distinguish CAT1-mediated effects from other transport mechanisms, researchers should implement these methodological approaches:

• Specific Inhibition Studies: Use competitive inhibitors of cationic amino acid transport (e.g., L-lysine, L-arginine) at varying concentrations to determine whether observed effects are specifically due to CAT1 transport activity.

• Gene Silencing with Multiple Controls: When performing CAT1 knockdown:

  • Use multiple siRNAs targeting different regions of the CAT1/SLC7A1 gene to minimize off-target effects

  • Include a negative control siRNA (e.g., Silencer select siRNA Negative Control Med GC) to account for non-specific effects of the transfection process

  • Quantify knockdown efficiency using Western blot analysis with anti-CAT1 antibody

  • Measure effects on multiple transport systems to identify specific versus general transport disruption

• Rescue Experiments: After CAT1 knockdown, perform rescue experiments by reintroducing:

  • Wild-type CAT1

  • Transport-deficient CAT1 mutants

  • Other cationic amino acid transporters (e.g., CAT2, CAT3)

  This approach helps determine whether the observed phenotype is specifically due to CAT1's transport function or other functions.

• Monitoring Multiple Endpoints: Simultaneously assess:

  • Amino acid uptake (using radiolabeled amino acids)

  • Downstream signaling pathways

  • Cell behavior (proliferation, migration)

  • Gene expression changes

  This comprehensive approach allows researchers to correlate CAT1 transport activity with specific cellular responses.

What are the optimal protocols for CAT1 detection using Western blot?

For optimal CAT1 detection using Western blot, researchers should follow these methodological steps:

• Sample Preparation:

  • For cell lysates: add protein extraction buffer with protease inhibitors

  • Include β-actin (1:1000, Santa Cruz) as a loading control

  • For quantitative comparisons, use ImageJ software to analyze densitometry of the corresponding proteins

• Antibody Selection and Dilution:

  • Primary antibody: Anti-CAT1 antibody (such as clone 2B9) at 1:500-1:1000 dilution

  • Secondary antibody: HRP-labeled appropriate species antibody (1:5000-1:10000)

  • Incubation: Primary antibody overnight at 4°C, secondary antibody for 1 hour at room temperature

• Detection System:

  • Use enhanced chemiluminescence (ECL) or Odyssey Infrared Imaging System for protein detection

  • Expected molecular weight for CAT1/SLC7A1: approximately 67.6 kDa

• Controls and Validation:

  • Positive control: SLC7A1-transfected cell lysate

  • Negative control: Non-transfected lysate

  • For knockdown validation: Compare CAT1 expression between control siRNA and CAT1 siRNA-treated samples

• Troubleshooting Tips:

  • If weak signal is observed, try longer exposure times or higher antibody concentration

  • If multiple bands appear, optimize blocking conditions (use 5% non-fat milk or BSA in TBST) and increase washing steps

  • For membrane proteins like CAT1, avoid excessive heating of samples which can cause aggregation

Following this protocol will help ensure specific and reproducible detection of CAT1 protein in experimental samples.

What methodology should be used for studying CAT1 knockdown effects?

For robust CAT1 knockdown studies, researchers should implement the following methodological approach:

• siRNA Design and Selection:

  • Design siRNAs targeting CAT1/SLC7A1 using specialized tools like GeneAssist Custom siRNA Builder

  • Use multiple siRNAs targeting different regions of the gene to confirm specificity

  • Include a negative control siRNA (e.g., Silencer select siRNA Negative Control Med GC)

• Transfection Protocol:

  • Transfect cells at 60-70% confluence to ensure efficient uptake

  • For adherent cells, use a lipid-based transfection reagent like Lipofectamine

  • Optimize siRNA concentration (typically 20-50 nM) to achieve maximum knockdown with minimal off-target effects

  • Incubate for 48-72 hours post-transfection before functional assays

• Knockdown Verification:

  • Protein Level: Perform Western blot analysis using anti-CAT1 antibody

  • mRNA Level: Conduct RT-qPCR with SLC7A1-specific primers, Power SYBR Green PCR Master Mix, and appropriate thermal cycling conditions (95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute)

  • Use appropriate housekeeping controls (β-actin for protein, TATA-binding protein for mRNA)

• Functional Assays Following Knockdown:

  • Transport Activity: Measure uptake of radiolabeled cationic amino acids

  • Cellular Phenotype: Assess changes in proliferation, migration, or other relevant cellular functions

  • BLV Binding: For viral studies, evaluate binding between CAT1 and BLV particles

  • Protein Interaction: Analyze changes in protein-protein interactions or downstream signaling pathways

• Data Analysis and Interpretation:

  • Normalize knockdown data to account for transfection efficiency

  • Perform statistical analysis to determine significance of observed changes

  • Consider dose-dependent and time-dependent effects of knockdown

This comprehensive approach ensures reliable assessment of CAT1's functional role in various cellular processes.

How can researchers optimize immunohistochemical detection of CAT1 in tissue samples?

For optimal immunohistochemical (IHC) detection of CAT1 in tissue samples, researchers should implement the following methodological protocol:

• Tissue Preparation:

  • Fix tissues in 10% formalin for 24-48 hours

  • Embed in paraffin and section at 5 μm thickness

  • For antigen retrieval, treat with citrate buffer (pH 6.0) for 20 minutes at 95°C

• Blocking and Antibody Incubation:

  • Block endogenous peroxidase activity with 0.3% H₂O₂ in methanol for 10 minutes

  • Block non-specific binding with 5% normal serum from the same species as the secondary antibody

  • Incubate with primary anti-CAT1 antibody at optimized concentration (typically 1:100-1:200) overnight at 4°C

  • Wash thoroughly with PBS containing 0.1% Tween-20

• Detection System:

  • For rat monoclonal antibodies: Use biotinylated rabbit anti-rat IgG (1:200) for 1 hour

  • Incubate with avidin-biotinylated enzyme Complex solution for 30 minutes

  • Develop with 0.05% 3,3'-diaminobenzidine and 0.01% H₂O₂ in 0.1 M Tris-HCl (pH 7.4)

  • Counterstain with hematoxylin

• Controls and Validation:

  • Positive Control: Include known CAT1-expressing tissue

  • Negative Control: Use isotype-matched control (e.g., rat IgG (γ2b/κ) for rat monoclonal antibodies)

  • Antibody Validation: Test antibody specificity on CAT1-knockdown tissues/cells

• Scoring and Interpretation:

  • Implement a standardized scoring system similar to the HER2 test

  • Score CAT1 expression as: 0 (negative), 1 (weak/borderline), 2 (intermediate), or 3 (strong)

  • Have at least two independent pathologists evaluate the staining

  • Compare expression between normal tissue (adjacent to or distant from disease site) and pathological tissue

This systematic approach ensures reliable and reproducible detection of CAT1 in tissue samples for both diagnostic and research applications.

What techniques are available for studying CAT1-virus interactions?

For studying CAT1-virus interactions, particularly with bovine leukemia virus (BLV), researchers can employ these specialized techniques:

• Binding Assays:

  • Incubate cells with fluorescently labeled viral particles

  • Analyze binding by flow cytometry to quantify virus attachment to cell surface

  • Compare binding in CAT1-expressing versus CAT1-negative cells

  • For competition assays, pre-incubate cells with anti-CAT1 antibodies to block binding sites

• Colocalization Studies:

  • Transfect cells with CAT1-GFP fusion constructs

  • Infect with fluorescently labeled viral particles

  • Perform confocal microscopy to visualize colocalization

  • Use DAPI (0.5 μg/mL) for nuclear staining

  • Analyze images using colocalization coefficients (Pearson's or Mander's)

• Syncytium Formation Assay:

  • Co-culture CAT1-expressing cells with BLV-infected cells

  • Monitor formation of multinucleated syncytia over time (typically 24-72 hours)

  • Quantify by counting the number of nuclei per syncytium or the percentage of nuclei in syncytia

  • This serves as a functional readout of BLV Env-CAT1 interaction

• Receptor Expression Modulation:

  • Generate stable CAT1-overexpressing cells using:

    • Human CAT1 cDNA obtained by RT of total RNA from cells (e.g., HT29)

    • Cloning into expression vectors (e.g., pcDNA3.1-EGFP)

    • Transfection using Lipofectamine 2000 or 293fectin

    • Selection with G418 (400 μg/mL) and sorting based on GFP expression

  • Alternatively, perform CAT1 knockdown using siRNA to assess the impact on viral infection

• Cross-Species CAT1 Analysis:

  • Express CAT1 from different species in CAT1-negative cells

  • Compare susceptibility to viral infection across species variants

  • This approach has revealed that CAT1 exhibits no species specificity for BLV infection, explaining its broad host range

These methodologies provide comprehensive insights into the mechanisms of CAT1-virus interactions and potential therapeutic targets for viral diseases.

What are common challenges in CAT1 antibody experiments and how can they be addressed?

Researchers frequently encounter several challenges when working with CAT1 antibodies. Here are the most common issues and recommended solutions:

• Variable Antibody Performance Across Applications:

  • Problem: An antibody may work well for Western blot but poorly for immunohistochemistry

  • Solution: Validate each antibody for specific applications; optimize conditions for each technique; consider using application-specific antibodies from manufacturers

• Cross-Reactivity Issues:

  • Problem: Antibodies may detect proteins other than CAT1

  • Solution: Verify specificity using CAT1 knockdown controls; compare results with multiple anti-CAT1 antibodies targeting different epitopes; include appropriate negative controls (isotype-matched antibodies)

• Detection in Membrane Preparations:

  • Problem: CAT1 is a transmembrane protein with 14 putative transmembrane domains, making extraction challenging

  • Solution: Use appropriate detergents (e.g., 1% Triton X-100 or RIPA buffer with 0.1% SDS); avoid excessive heating of samples; consider membrane-enriched fractionation techniques

• Variability in Signal Intensity:

  • Problem: Inconsistent detection levels between experiments

  • Solution: Standardize protein loading (verified by housekeeping proteins like β-actin); maintain consistent antibody concentrations and incubation times; prepare fresh reagents for each experiment

• Background Issues in Immunohistochemistry:

  • Problem: High background staining obscuring specific signals

  • Solution: Optimize blocking conditions (use 5% serum from the same species as secondary antibody); extend washing steps; titrate primary antibody concentration; include appropriate negative controls (tissues from CAT1-knockout models or isotype controls)

• Interpreting Knockdown Efficiency:

  • Problem: Incomplete CAT1 knockdown leading to ambiguous results

  • Solution: Use multiple siRNAs targeting different regions; quantify knockdown at both protein and mRNA levels; establish dose-response relationships for functional readouts relative to knockdown efficiency

By implementing these troubleshooting approaches, researchers can enhance the reliability and reproducibility of their CAT1 antibody experiments.

How should researchers interpret CAT1 expression data in the context of cancer studies?

When interpreting CAT1 expression data in cancer research contexts, researchers should consider these critical factors:

• Expression Level Thresholds:

  • Implement standardized scoring systems (0-3 scale) similar to established clinical markers like HER2

  • Define clear cutoff values that distinguish between negative, weak, intermediate, and strong expression

  • For quantitative techniques (Western blot, qPCR), normalize to appropriate housekeeping genes and establish fold-change thresholds

• Cellular Localization Patterns:

  • Assess whether CAT1 is primarily localized to cell membrane, cytoplasm, or shows altered distribution in cancer cells

  • Correlate subcellular localization with functional outcomes (e.g., membrane localization is critical for transport function)

  • Note that internalization patterns may differ between normal and cancer cells, potentially reflecting altered trafficking

• Correlation with Clinical Parameters:

  • Analyze CAT1 expression in relation to:

    • Tumor stage and grade

    • Metastatic potential

    • Patient survival outcomes

    • Treatment response

  • Overexpression in >70% of human CRC samples suggests potential value as a biomarker, but requires correlation with these clinical parameters

• Integration with Other Molecular Markers:

  • Examine CAT1 expression alongside other cancer-related proteins (e.g., HIF-1α)

  • Consider analyzing expression in the context of regulatory microRNAs (e.g., miRNA214, miRNA378)

  • Integrate with data on chromosomal aberrations, particularly amplifications at 13q12.3 region

• Functional Validation:

  • Support expression data with functional assays:

    • Assess impact of CAT1 knockdown on cancer cell proliferation

    • Measure effects of anti-CAT1 antibodies on cell migration and growth

    • Evaluate in vivo tumor growth in models with modulated CAT1 expression

• Heterogeneity Considerations:

  • Account for intratumoral heterogeneity by examining multiple regions within tumors

  • Compare expression between primary tumors and metastatic sites

  • Evaluate expression across different patient cohorts and cancer subtypes

This comprehensive analytical approach ensures robust interpretation of CAT1 expression data in cancer research, facilitating its potential application as both a biomarker and therapeutic target.

What controls are essential when evaluating antibody specificity for CAT1 detection?

To ensure reliable evaluation of antibody specificity for CAT1 detection, researchers must implement these essential controls:

• Genetic Controls:

  • Positive Control: Cells overexpressing CAT1 (e.g., CAT1-transfected 293T or HEK293F cells)

  • Negative Control: CAT1-negative cell lines (e.g., CHO-K1 cells)

  • Knockdown Control: Cells treated with CAT1-specific siRNA compared to non-targeting siRNA

  • Cross-species Controls: When testing antibody across species, include species-specific positive and negative controls

• Antibody Controls:

  • Isotype Control: Use isotype-matched antibodies (e.g., rat IgG (γ2b/κ) for rat monoclonal antibodies) at the same concentration as the CAT1 antibody

  • Concentration Gradient: Test antibody specificity across a range of concentrations to determine optimal signal-to-noise ratio

  • Multiple Antibodies: When possible, verify results using antibodies targeting different epitopes of CAT1

• Method-Specific Controls:

  • Western Blot:

    • Loading control (β-actin) to normalize protein amounts

    • Molecular weight markers to confirm band size (expected 67.6 kDa for CAT1)

    • Secondary antibody-only control to detect non-specific binding

  • Immunohistochemistry/Immunofluorescence:

    • Secondary antibody-only control

    • Known positive and negative tissue sections

    • Blocking peptide competition (pre-incubating antibody with excess CAT1 peptide)

  • Flow Cytometry:

    • Unstained cells to establish baseline autofluorescence

    • Secondary antibody-only control

    • Isotype-matched control antibody

• Validation Across Methods:

  • Confirm CAT1 detection using complementary techniques:

    • Protein detection (Western blot, IHC, flow cytometry)

    • mRNA quantification (RT-qPCR)

    • Functional assays (amino acid transport)

By implementing this comprehensive set of controls, researchers can confidently establish antibody specificity and ensure reliable detection of CAT1 across experimental contexts and applications.