ERCC6L Antibody

What is ERCC6L and why is it significant in cancer research?

ERCC6L is a recently discovered DNA helicase that has been demonstrated to be highly expressed in various human cancer types, including colorectal cancer, gastric cancer, breast cancer, and lung adenocarcinoma. Its significance lies in its role in promoting cancer cell growth, invasion, and metastasis, making it a potential biomarker and therapeutic target. Studies have shown that ERCC6L knockdown significantly inhibits proliferation and colony-forming ability of cancer cell lines while decreasing invasion capability .

How is ERCC6L expression typically analyzed in tumor samples?

ERCC6L expression is commonly analyzed through multiple complementary techniques. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used to quantify mRNA expression levels, while western blot analysis determines protein abundance. Immunohistochemistry (IHC) allows for visualization of ERCC6L localization within tissue samples, revealing that ERCC6L is expressed in both the cytoplasm and nucleus of cells, with cytoplasmic expression typically higher than nuclear expression . These multi-modal approaches provide comprehensive characterization of ERCC6L expression patterns.

What are the typical controls used when evaluating ERCC6L expression in cancer tissues?

When evaluating ERCC6L expression in cancer tissues, researchers typically use matched pairs of cancerous and adjacent noncancerous tissues from the same patients as controls. For cell line studies, normal cell lines corresponding to the tissue of origin are used as controls (e.g., normal colonic mucosal cell line NCM460 for colorectal cancer studies or GES-1 for gastric cancer studies) . These paired comparisons allow for direct assessment of ERCC6L upregulation in the malignant state.

How does ERCC6L influence the cell cycle in cancer cells?

ERCC6L appears to accelerate the cell cycle by regulating the G2/M checkpoint signaling pathway. Flow cytometric analysis has demonstrated that ERCC6L knockdown in colorectal cancer cells inhibits cell cycle progression and increases the number of cells in the G0/G1 phase without affecting apoptosis . In breast cancer, ERCC6L has been shown to interact with KIF4A, a factor closely related to mitosis, further supporting its role in cell cycle regulation . This suggests that ERCC6L may promote cancer progression by overcoming cell cycle checkpoints.

What signaling pathways does ERCC6L interact with in different cancer types?

The signaling mechanisms of ERCC6L appear to vary across cancer types. In gastric cancer, ERCC6L overexpression increases phosphorylated NF-κB p65 levels, suggesting involvement of the NF-κB pathway . In breast cancer, ERCC6L accelerates the cell cycle by regulating the G2/M checkpoint signaling pathway . There are also indications from other studies (though not in the provided search results) that ERCC6L may involve the PI3K/AKT pathway in hepatocellular carcinoma. This suggests context-dependent mechanisms of action that may require cancer-specific investigation.

What is the relationship between ERCC6L and epithelial-mesenchymal transition (EMT)?

ERCC6L appears to promote EMT, a critical process in cancer metastasis. In gastric cancer cells, ERCC6L overexpression increases N-cadherin expression while decreasing E-cadherin expression - classic markers of EMT. Conversely, ERCC6L knockdown reverses these effects . This modulation of adhesion molecules explains in part how ERCC6L enhances migration and invasion capabilities of cancer cells, contributing to metastatic potential.

What are the optimal fixation protocols for immunohistochemical detection of ERCC6L?

For immunohistochemical detection of ERCC6L, researchers typically use 4% paraformaldehyde (PFA) fixation for approximately 20 minutes for cell preparations, followed by permeabilization with 0.3% Triton X for 10 minutes and blocking with 5% bovine serum albumin (BSA) for 30 minutes at room temperature . For tissue samples, standard formalin fixation and paraffin embedding protocols are employed. Primary ERCC6L antibody concentrations of 1:100 dilution are commonly used with overnight incubation at 4°C to achieve optimal staining results.

What is the recommended approach for ERCC6L knockdown experiments?

ERCC6L knockdown experiments typically employ small interfering RNA (siRNA) or short hairpin RNA (shRNA) approaches. When using siRNA, researchers often test multiple siRNA constructs to identify the most effective sequence. For example, in colorectal cancer studies, three different siRNAs were tested, with si-ERCC6L-103 providing the greatest reduction in expression . For stable knockdown, lentiviral shRNA constructs are used followed by selection with puromycin (typically 3 μg/mL) . Knockdown efficiency should be validated at both mRNA and protein levels through RT-qPCR and western blot.

What are the best experimental designs to study ERCC6L's effect on cell migration and invasion?

The most robust approach to study ERCC6L's effect on cell migration and invasion involves a combination of complementary assays. Wound healing (scratch) assays provide qualitative and semi-quantitative assessment of migration capability, while Transwell assays with or without Matrigel coating quantitatively measure invasion and migration, respectively . These should be complemented with proliferation assays (MTT or similar) to distinguish between effects on motility versus growth. Additionally, measuring EMT markers through western blot provides mechanistic insight into migration and invasion phenotypes.

How should researchers account for cell line-specific differences in ERCC6L function?

Researchers should be aware that ERCC6L effects may vary between cell lines due to genetic background differences. For example, in colorectal cancer studies, the ability to migrate, invade, and form colonies was higher in SW480 cells compared to HT29 cells after ERCC6L knockdown, and significant cell cycle arrest at G0/G1 was only observed in HT29 cells . These differences may result from variations in genetic background, such as KRAS and BRAF mutations, which affect downstream signaling pathways. Using multiple cell lines representing different molecular subtypes is recommended for comprehensive characterization.

What approaches can resolve discrepancies between mRNA and protein levels of ERCC6L?

When discrepancies arise between mRNA and protein levels of ERCC6L, researchers should employ multiple strategies to resolve them. First, evaluate mRNA stability through actinomycin D chase experiments to determine if post-transcriptional regulation is occurring. Second, assess protein stability using cycloheximide chase assays to identify potential differences in protein turnover. Third, investigate potential microRNA regulation by correlating expression of predicted regulatory microRNAs with ERCC6L levels. Finally, consider technical factors like antibody specificity and the sensitivity differences between RT-qPCR and western blot methods.

How can researchers differentiate between direct and indirect effects of ERCC6L on cellular phenotypes?

To differentiate between direct and indirect effects of ERCC6L, researchers should employ rescue experiments where ERCC6L expression is restored in knockdown cells to confirm phenotype reversal. Co-immunoprecipitation and proximity ligation assays can identify direct protein-protein interactions, such as the reported interaction between ERCC6L and KIF4A . Chromatin immunoprecipitation (ChIP) can determine if ERCC6L directly regulates gene expression. Time-course experiments following ERCC6L manipulation can help establish the sequence of events, while pathway inhibitors can clarify which downstream effectors mediate ERCC6L's effects.

What are the considerations for designing a transgenic mouse model to study ERCC6L function?

When designing a transgenic mouse model to study ERCC6L function, researchers should consider using conditional knockout approaches, as seen in the ERCC6L flox/flox system crossed with tissue-specific Cre lines like MMTV-Cre for mammary gland studies . This approach allows for tissue-specific deletion while avoiding potential embryonic lethality if ERCC6L is essential for development. The model should include appropriate controls, including littermates lacking Cre recombinase. Phenotypic characterization should assess both normal tissue development and cancer formation through careful monitoring protocols, with comprehensive histopathological analysis.

What experimental approaches best elucidate the interaction between ERCC6L and other mitotic proteins?

To elucidate interactions between ERCC6L and other mitotic proteins such as KIF4A, researchers should employ a multi-faceted approach. Immunofluorescence co-localization using confocal microscopy can demonstrate spatial proximity . Co-immunoprecipitation followed by mass spectrometry can identify interaction partners in an unbiased manner. Proximity ligation assays provide higher resolution evidence of protein-protein interactions in situ. FRET (Förster Resonance Energy Transfer) or BRET (Bioluminescence Resonance Energy Transfer) approaches can provide dynamic information about interactions in living cells. These methods collectively build a comprehensive picture of ERCC6L's interaction network.

What strategies can address non-specific binding issues with ERCC6L antibodies?

To address non-specific binding with ERCC6L antibodies, researchers should optimize blocking conditions using 5% BSA or 5-10% normal serum from the species in which the secondary antibody was raised. Increasing washing stringency with PBS-T (0.1-0.3% Tween-20) may reduce background. Pre-adsorption of antibodies with recombinant ERCC6L protein can confirm specificity. Using ERCC6L-knockout or knockdown samples as negative controls validates antibody specificity. Testing multiple commercial antibodies targeting different epitopes can identify the most specific reagent. For western blots, including peptide competition controls helps confirm band specificity.

How can researchers validate the specificity of ERCC6L antibodies in their experimental system?

Researchers should validate ERCC6L antibody specificity through multiple approaches. First, perform immunoblotting with recombinant ERCC6L protein alongside cell/tissue lysates to confirm the correct molecular weight. Second, use ERCC6L-knockdown or knockout samples as negative controls to demonstrate signal reduction. Third, test specificity in immunoprecipitation followed by mass spectrometry to confirm ERCC6L enrichment. Fourth, employ immunofluorescence in cells with modulated ERCC6L expression to verify localization patterns. Finally, compare results from multiple antibodies targeting different ERCC6L epitopes to ensure consistency in detection patterns.

What controls should be included when studying ERCC6L interactions with other proteins?

When studying ERCC6L interactions with other proteins, essential controls include reverse immunoprecipitation (pulling down the suspected interacting protein and blotting for ERCC6L) to confirm bidirectional interaction. IgG controls from the same species as the primary antibody are crucial to identify non-specific binding. Lysate input controls (typically 5-10% of immunoprecipitation input) establish baseline protein levels. RNase and DNase treatments during immunoprecipitation can differentiate between direct protein interactions versus co-association on nucleic acids. Competition with recombinant proteins can confirm specificity of observed interactions.