CAV1 (Ab-14) Antibody

What is CAV1 (Ab-14) Antibody and what epitope does it recognize?

CAV1 (Ab-14) Antibody is a polyclonal antibody raised in rabbits that specifically recognizes a peptide sequence around amino acids 12-16 (H-L-Y-T-V) derived from human caveolin-1 . This region contains the critical tyrosine-14 residue, which is a major phosphorylation site that regulates caveolin-1 function. The antibody is designed to detect both phosphorylated and non-phosphorylated forms of caveolin-1, depending on the specific clone. For phosphorylation-specific detection, researchers should use phospho-specific antibodies like those that specifically recognize phosphorylated tyrosine-14 .

What applications is CAV1 (Ab-14) Antibody validated for?

The CAV1 (Ab-14) Antibody has been validated for several laboratory applications including:

For Western blotting, the antibody reliably detects caveolin-1 at the expected molecular weights of 23-25 kDa, representing different isoforms and post-translational modifications .

What species reactivity does CAV1 (Ab-14) Antibody demonstrate?

The CAV1 (Ab-14) Antibody demonstrates cross-reactivity with multiple species:

• Human (primary validated target)

• Mouse

• Rat

This multi-species reactivity makes the antibody valuable for comparative studies across different model systems. The cross-reactivity stems from the high conservation of the target epitope sequence (H-L-Y-T-V) across these mammalian species .

How can I evaluate CAV1 phosphorylation at Tyrosine-14 in cancer cells?

To evaluate CAV1 phosphorylation at Tyrosine-14 in cancer cells, a multi-methodological approach is recommended:

• Western Blotting: Use phospho-specific antibodies that recognize CAV1 only when phosphorylated at Tyr14. Run parallel blots with total CAV1 antibodies to normalize phosphorylation levels .

• Immunoprecipitation: Immunoprecipitate CAV1 using a total CAV1 antibody, then probe with phospho-tyrosine antibodies or phospho-specific CAV1 (Tyr14) antibodies .

• Phosphatase Controls: Treat samples with phosphatases prior to analysis as negative controls to confirm phospho-specificity. For example, in the PTPN14 studies, researchers used phosphatase treatment to validate the specificity of tyrosine-14 phosphorylation signals .

• Stimulus Response: Evaluate changes in phosphorylation following treatments known to modulate CAV1 phosphorylation (e.g., growth factors, stress conditions) to confirm the functionality of the phosphorylation site .

For quantification, normalize phospho-CAV1 signal to total CAV1 to account for variations in total protein expression levels between samples .

What is the significance of CAV1 Y14 phosphorylation in cancer metastasis?

CAV1 phosphorylation at tyrosine-14 (pY14) serves as a molecular switch that promotes cancer cell migration, invasion, and metastasis:

• Rac-1 Activation: CAV1 pY14 facilitates the activation of the small GTPase Rac-1 in colon, breast, and melanoma cancer cells, enhancing cell migration and invasion abilities .

• Metastatic Potential: In vivo studies using B16F10 melanoma cells demonstrated that CAV1 expression increased metastasis approximately threefold compared to controls, and this effect was dependent on Y14 phosphorylation .

• Regulatory Mechanisms: The metastasis-promoting effects of phosphorylated CAV1 can be suppressed by:

  • E-cadherin co-expression, which forms a multiprotein complex with CAV1

  • PTPN14 phosphatase activity, which directly dephosphorylates CAV1 at Y14

• Signaling Pathway: CAV1 pY14 activates a signaling axis involving Rab-5 through sequestration of the GAP protein p85α, which promotes Tiam1 recruitment to early endosomes and subsequent Rac-1 activation .

These findings suggest that targeting CAV1 Y14 phosphorylation could be a potential therapeutic strategy for reducing cancer metastasis.

How does PTPN14 interact with CAV1 and what are the functional consequences?

PTPN14 (protein tyrosine phosphatase non-receptor type 14) has been identified as a novel regulator of CAV1 function through direct dephosphorylation:

• Complex Formation: Mass spectrometry analysis revealed that PTPN14 exists in a multiprotein complex with CAV1 and E-cadherin. This complex formation is facilitated by E-cadherin but can also occur directly between PTPN14 and CAV1, particularly with the CAV1(Y14F) mutant .

• Enzymatic Activity: PTPN14 directly dephosphorylates CAV1 at tyrosine-14, as demonstrated through co-immunoprecipitation experiments and phosphorylation analysis. The catalytic activity of PTPN14 was required for this dephosphorylation, as shown using domain deletion mutants .

• Functional Outcomes:

  • Reduced CAV1 phosphorylation on tyrosine-14

  • Suppressed CAV1-enhanced cell migration and invasion

  • Inhibited Rac-1 activation

  • Decreased metastasis in vivo

• Domain Requirements: The catalytic phosphatase domain of PTPN14 is essential for inhibiting CAV1-induced migration and invasion, while the N-terminal FERM domain contributes to, but is not essential for, these effects .

These findings identify CAV1 as a novel substrate for PTPN14 and demonstrate that PTPN14 expression is sufficient to counteract the metastasis-promoting effects of CAV1 through dephosphorylation of tyrosine-14.

What are the optimal conditions for Western blotting with CAV1 (Ab-14) Antibody?

For optimal Western blotting results with CAV1 (Ab-14) Antibody, follow these protocol recommendations:

• Sample Preparation:

  • Use RIPA or NP-40 based lysis buffers containing phosphatase inhibitors (crucial for preserving phosphorylation status)

  • Load 10-30 μg of total protein per lane

  • Include both reducing and denaturing conditions (with SDS and β-mercaptoethanol)

• Gel and Transfer Parameters:

  • Use 10-12% polyacrylamide gels due to the relatively small size of CAV1 (23-25 kDa)

  • Transfer to PVDF membranes (preferred over nitrocellulose for phosphorylated proteins)

• Antibody Incubation:

  • Primary antibody dilution: 1:500-1:1000 in 5% BSA in TBST

  • Incubation: Overnight at 4°C

• Expected Results:

  • Molecular weight: 23 kDa (α-isoform) and/or 25 kDa (β-isoform)

  • Phosphorylated CAV1 appears primarily at 25 kDa

• Buffer Composition:

  • The antibody is supplied in phosphate buffered saline without Mg2+ and Ca2+, pH 7.4, 150mM NaCl, 0.02% sodium azide, and 50% glycerol

For phospho-specific detection, ensure complete suppression of phosphatase activity during all preparation steps and consider comparing results with and without phosphatase treatment as controls.

How can I troubleshoot weak or non-specific signals when using CAV1 (Ab-14) Antibody?

When encountering issues with CAV1 (Ab-14) Antibody detection, consider these troubleshooting approaches:

• For Weak Signals:

  • Increase antibody concentration (try 1:250 instead of 1:1000)

  • Extend primary antibody incubation time to 48 hours at 4°C

  • Enhance detection using more sensitive substrates (e.g., femto-level ECL)

  • Enrich for membrane fractions during sample preparation as CAV1 is a membrane protein

• For Non-specific Bands:

  • Optimize blocking conditions (try 5% BSA instead of milk for phospho-detection)

  • Include a CAV1 knockout or knockdown sample as a negative control

  • Use more stringent washing conditions (increase wash times and detergent concentration)

  • Consider using antibodies purified by affinity-chromatography using epitope-specific peptides

• For Phosphorylation-specific Issues:

  • Ensure all buffers contain fresh phosphatase inhibitors

  • Use positive controls (cells treated with pervanadate to enhance tyrosine phosphorylation)

  • Compare phospho-specific signals with total CAV1 detection

  • Include phosphatase-treated samples as controls

• Sample Handling Issues:

  • Store antibody at -20°C or -80°C and avoid repeated freeze-thaw cycles

  • Freshly prepare protein samples and avoid storage when examining phosphorylation status

The antibody has been shown to reliably detect endogenous levels of CAV1 when these conditions are properly optimized .

How can CAV1 (Ab-14) Antibody be used to study cancer cell migration and invasion?

CAV1 (Ab-14) Antibody can be strategically employed in multiple experimental setups to investigate cancer cell migration and invasion processes:

• Correlation Studies:

  • Assess the relationship between CAV1 Y14 phosphorylation levels and migration/invasion capacity across different cancer cell lines

  • Perform Western blotting with phospho-CAV1 (Y14) antibodies and correlate with results from migration/invasion assays

• Intervention Experiments:

  • Compare wild-type CAV1 with phosphorylation-deficient mutants (CAV1-Y14F)

  • Assess the impact of phosphatase overexpression (e.g., PTPN14) on CAV1 phosphorylation and cell migration

  • Examine the effects of E-cadherin expression on CAV1 phosphorylation status and function

• Mechanistic Studies:

  • Use immunoprecipitation with CAV1 (Ab-14) Antibody to identify novel interaction partners in migrating cells

  • Perform immunofluorescence to visualize the subcellular localization of phosphorylated CAV1 during migration

  • Assess Rac-1 and Rab-5 activation in relation to CAV1 phosphorylation status

• In Vivo Applications:

  • Generate stable cell lines with varying levels of CAV1 and CAV1-Y14 phosphorylation

  • Inject these cells into animal models to assess metastatic potential

  • Use tissue samples to analyze CAV1 phosphorylation in primary tumors versus metastatic lesions

Research has demonstrated that CAV1 phosphorylation at Y14 significantly enhances migration, invasion, and metastasis, making this antibody valuable for studying these processes in cancer research .

Can CAV1 (Ab-14) Antibody be used to study the interaction between CAV1 and E-cadherin?

Yes, CAV1 (Ab-14) Antibody can be effectively used to study the interaction between CAV1 and E-cadherin, which has significant implications for cancer metastasis:

• Co-immunoprecipitation:

  • Use CAV1 (Ab-14) Antibody to immunoprecipitate CAV1 complexes

  • Detect E-cadherin in the immunoprecipitates by Western blotting

  • This approach has successfully demonstrated that CAV1 and E-cadherin form a multiprotein complex that includes β-catenin and PTPN14

• Reciprocal Co-immunoprecipitation:

  • Immunoprecipitate E-cadherin and detect CAV1 in the precipitates

  • Compare complex formation in different cancer cell lines with varying metastatic potential

• Proximity Ligation Assay:

  • Use CAV1 (Ab-14) Antibody in combination with E-cadherin antibodies to visualize direct protein-protein interactions in situ

  • This technique can reveal the subcellular localization of these interactions

• Functional Assays:

  • Assess how E-cadherin expression affects CAV1 Y14 phosphorylation using phospho-specific antibodies

  • Determine whether E-cadherin-mediated recruitment of PTPN14 to CAV1 affects downstream signaling pathways

  • Investigate how this interaction modulates Rac-1 activation and cancer cell migration/invasion

Research has shown that E-cadherin co-expression inhibits CAV1-enhanced migration, invasion, and metastasis by facilitating the recruitment of PTPN14, which dephosphorylates CAV1 at Y14 .

How should I interpret multiple bands in Western blot using CAV1 (Ab-14) Antibody?

When analyzing Western blots with CAV1 (Ab-14) Antibody, multiple bands may appear that require careful interpretation:

• Expected Bands:

  • 23 kDa: α-isoform of CAV1

  • 25 kDa: β-isoform of CAV1 or phosphorylated α-isoform

• Common Variations:

  • Phosphorylated CAV1 typically appears as a slightly higher molecular weight band compared to unphosphorylated form

  • The presence/absence of multiple bands may vary by cell type and activation state

  • Treatment with phosphatases should eliminate phosphorylation-specific bands

• Potential Artifacts:

  • Bands above 30 kDa: May represent CAV1 oligomers or complexes that weren't fully denatured

  • Bands below 20 kDa: Could indicate proteolytic degradation products

  • Non-specific bands: Verify with appropriate controls (knockdown/knockout samples)

• Quantification Approach:

  • For total CAV1: Quantify all specific CAV1 bands (typically 23-25 kDa)

  • For phosphorylation studies: Calculate the ratio of phosphorylated to total CAV1

  • Use appropriate normalization with loading controls (β-actin, GAPDH)

For accurate interpretation, always run appropriate controls including phosphatase-treated samples when studying phosphorylation and validate band specificity with knockdown experiments when possible.

What is the biological significance of CAV1's role in the PTPN14/CAV1/E-cadherin axis?

The biological significance of the PTPN14/CAV1/E-cadherin regulatory axis represents a novel mechanism of metastasis control with important implications:

• Metastasis Suppression Mechanism:

  • E-cadherin, often downregulated in metastatic cancer, forms a complex with CAV1

  • This complex recruits PTPN14, which dephosphorylates CAV1 at tyrosine-14

  • Dephosphorylation prevents CAV1 from activating Rac-1, thereby inhibiting migration, invasion, and metastasis

• Quantitative Impact:

  • Expression of CAV1 alone increases metastasis approximately threefold in experimental models

  • Co-expression of E-cadherin or PTPN14 reduces CAV1-induced metastasis to levels comparable to control cells

  • Overexpression of PTPN14 in B16F10(CAV1) cells reduced metastatic lung mass from 35.5% to 1.6%

• Molecular Pathway Model:

  • Without E-cadherin: CAV1 phosphorylation on Y14 → Rac-1 activation → enhanced migration/invasion

  • With E-cadherin: Multiprotein complex formation → PTPN14 recruitment → CAV1-Y14 dephosphorylation → inhibition of Rac-1 activation

• Clinical Implications:

  • Lower PTPN14 expression (threefold reduction) has been observed in invasive ductal breast carcinomas compared to ductal carcinomas

  • Survival of breast cancer patients decreased if tumors expressed high levels of PTPN14 substrates

  • This pathway represents a potential therapeutic target for metastasis prevention in cancers with high CAV1 expression

This regulatory axis provides insight into how the tumor suppressor functions of E-cadherin and PTPN14 converge to control CAV1-mediated signaling in cancer progression .