Phospho-CLDN7 (Tyr210) Antibody

What is Claudin-7 and its phosphorylated form at Tyrosine 210?

Claudin-7 (CLDN7) is a member of the claudin family of integral membrane proteins that form tight junction strands between epithelial or endothelial cells. These tight junctions serve as physical barriers preventing solutes and water from passing freely through the paracellular space, while also maintaining cell polarity and mediating signal transduction .

Phosphorylation at Tyrosine 210 (Tyr210) represents a critical post-translational modification that regulates Claudin-7 function. This specific phosphorylation site is located near the C-terminal region of the protein (amino acids 162-211) and affects tight junction integrity and paracellular permeability .

What experimental applications are supported by Phospho-CLDN7 (Tyr210) antibodies?

Phospho-CLDN7 (Tyr210) antibodies are validated for multiple research applications:

Most commercially available antibodies are rabbit polyclonal antibodies with reactivity to human, mouse, and rat samples .

How should researchers optimize detection of Phospho-CLDN7 (Tyr210) in Western blot experiments?

For optimal Western blot detection:

• Sample preparation: Use fresh tissue or cell lysates with phosphatase inhibitors to preserve phosphorylation states

• Protein loading: Load 20-30 μg of total protein per lane

• Gel percentage: Use 5-20% SDS-PAGE gels for optimal separation

• Transfer conditions: Transfer at 150 mA for 50-90 minutes to nitrocellulose membrane

• Blocking: Block with 5% non-fat milk in TBS for 1.5 hours at room temperature

• Primary antibody: Incubate with anti-Phospho-CLDN7 (Tyr210) antibody (0.25-1 μg/ml) overnight at 4°C

• Washing: Wash with TBS-0.1% Tween three times, 5 minutes each

• Secondary antibody: Use HRP-conjugated anti-rabbit IgG at 1:5000 dilution

• Detection: Develop using enhanced chemiluminescence (ECL)

• Controls: Include non-phosphorylated controls and antigen-specific peptide competition as shown in research studies

What is known about the tissue distribution of Phospho-CLDN7 (Tyr210)?

Immunohistochemical studies have revealed that phosphorylated Claudin-7 exhibits a specific distribution pattern in tissues:

• In colonic epithelium: Primarily localized at the tip of crypts and in the luminal colonic epithelium

• Cell membrane localization: Predominantly at the basolateral cell membrane and tight junctions

• Co-localization: Often co-localizes with EpCAM at the basolateral cell membrane

The phosphorylation status of Claudin-7 varies depending on tissue type, physiological state, and in disease conditions like inflammatory bowel disease and cancer .

How does LPS treatment affect Claudin-7 phosphorylation dynamics?

Research has revealed time-dependent changes in Claudin-7 phosphorylation in response to lipopolysaccharide (LPS) treatment:

The decrease in phosphorylated Claudin-7 at 72h after LPS treatment contrasts with other claudins - phosphorylated Claudin-6 increased at 72h, while phosphorylated Claudin-3 increased at 48h but decreased at 72h . This differential regulation suggests distinct roles for various claudin phosphorylation events during inflammatory responses.

Methodological approach: To study these dynamics, researchers should design time-course experiments with LPS treatment (typically 50-100 ng/ml) of colonic epithelial cells, followed by western blot analysis using phospho-specific antibodies for different claudin family members .

What is the relationship between Claudin-7 phosphorylation and paracellular permeability?

Enhanced phosphorylation of Claudin-7 has been associated with increased paracellular permeability and reduced tight junction strength . Multiple studies have demonstrated this relationship:

• In experimental colitis models, increased phosphorylated Claudin-7 content correlated with disrupted epithelial barrier function

• Studies using FITC-dextran flux assays showed that altered Claudin-7 phosphorylation affects paracellular permeability

• Electron microscopy revealed fuzzy tight junction structure in conditions with aberrant Claudin-7 phosphorylation

• Claudin-7 knockdown increased DSS-induced inflammatory injury and epithelial barrier disruption in vitro

Specifically, Claudin-7 phosphorylation affects the ratio of permeability of Cl⁻ to Na⁺, suggesting a role in ion selectivity of the paracellular pathway .

For experimental assessment: Researchers should employ Ussing chamber experiments to measure transepithelial electrical resistance (TEER), dilution potential, and ion flux measurements in combination with phosphorylation status analysis .

How does Claudin-7 knockout or deficiency affect WNK4 signaling pathways?

Claudin-7 knockout (CLDN7⁻/⁻) leads to significant alterations in WNK4 signaling pathways:

• Increased expression levels: CLDN7⁻/⁻ cells show significantly elevated mRNA and protein levels of WNK4, SGK-1, and SPAK compared to wild-type cells

• Ion channel modulation: ENaC-α, -β, and -γ subunits all exhibited increased expression in CLDN7⁻/⁻ cells, while ROMK and Na-K-ATPase remained unchanged

• Protein complex formation: Claudin-7 normally colocalizes with WNK4 in kidneys and forms a protein complex when co-expressed in kidney epithelial cells

This suggests that Claudin-7, particularly its phosphorylation state, plays a regulatory role in ion homeostasis through interaction with the WNK4/SPAK/SGK-1 signaling axis.

Experimental approach: Researchers can use CLDN7 knockdown models with shRNA, followed by rescue experiments to verify phenotype reversion. Real-time RT-PCR and immunoblotting should be performed to assess changes in expression levels of WNK4, SGK-1, SPAK, and associated ion channels .

What is the connection between p53/HNF4α and Claudin-7 phosphorylation?

Research indicates a complex regulatory relationship between transcription factors p53 and HNF4α and Claudin-7 expression/phosphorylation:

• Nuclear levels of p53 and HNF4α correlate with changes in Claudin-7 expression during cell differentiation

• Tenovin-1 (TEN), a p53 activator, increases nuclear levels of p53 and HNF4α, which in turn increases Claudin-7 mRNA levels and promoter activity

• p53 and HNF4α form a complex that binds to the promoter region of Claudin-7, enhancing its expression

• This p53/HNF4α regulation appears to be tissue-specific, with high expression at the surface of mouse colon crypt

This transcriptional regulation likely affects phosphorylation status as well, suggesting a coordinated regulation of both expression and post-translational modification.

For studying this regulatory network: Researchers should use chromatin immunoprecipitation (ChIP) assays to assess p53 and HNF4α binding to the Claudin-7 promoter, while employing phospho-specific antibodies to monitor how these transcriptional changes affect phosphorylation status .

What role does Phospho-CLDN7 (Tyr210) play in inflammatory bowel disease and colitis-associated cancer?

Claudin-7 phosphorylation status has emerged as a critical factor in inflammatory bowel disease (IBD) and colitis-associated cancer (CAC):

• Increased phosphorylation: Phosphorylated Claudin-7 content increases during experimental colitis, particularly at days 6 and 8

• Barrier dysfunction: This phosphorylation correlates with disrupted intestinal epithelial barrier integrity

• Inflammatory cascade: Cldn-7 deficiency promotes colitis by destroying tight junction integrity and increasing inflammatory cytokine production (IL-6, IL-8)

• Malignant transformation: Intestinal conditional Claudin-7 knockout mice spontaneously develop atypical hyperplasia and adenomas, indicating a tumor-suppressive role

• Prognostic value: Claudin-7 expression in colorectal cancer tissue correlates with tumor differentiation grade and may serve as a prognostic marker for disease-free survival

Methodological approach for research: Utilize dextran sodium sulfate (DSS) colitis models and azoxymethane (AOM)/DSS colorectal cancer models with Claudin-7 conditional knockout mice. Monitor phosphorylation status using phospho-specific antibodies while assessing barrier function through permeability assays and inflammatory markers through qRT-PCR and ELISA .

What are the best methodological approaches for studying in vitro phosphorylation of Claudin-7?

For studying Claudin-7 phosphorylation in vitro, researchers should consider these methodological approaches:

• Cell-Based ELISA: Provides a convenient, lysate-free, high-throughput method to detect Claudin-7 phosphorylation and expression profiles in intact cells

  • Allows for screening effects of various treatments, inhibitors, or activators

  • Can simultaneously measure total and phosphorylated Claudin-7 levels

  • Suitable for >5000 cells/well with colorimetric detection at 450 nm

• Phosphopeptide mapping:

  • Use synthesized phosphopeptides derived from human Claudin-7 around Tyr210 (S-K-E-YP-V) for antibody validation and as controls

  • Mass spectrometry-based approaches can identify novel phosphorylation sites

• Kinase/phosphatase identification:

  • Screen kinase inhibitors to identify enzymes responsible for Tyr210 phosphorylation

  • Use phosphatase inhibitors during sample preparation to preserve phosphorylation state

• Mutation studies:

  • Generate Tyr210-to-Phe (Y210F) or Tyr210-to-Glu (Y210E) mutants to create phospho-dead or phosphomimetic versions of Claudin-7

  • Express these mutants in CLDN7-knockout backgrounds to assess functional consequences

• Co-immunoprecipitation:

  • Use phospho-specific antibodies to identify interaction partners specific to the phosphorylated form

  • Compare interactomes between phosphorylated and non-phosphorylated Claudin-7

These approaches provide complementary information about the regulation and function of Claudin-7 phosphorylation at Tyrosine 210.