xpo6 Antibody

What is XPO6 and what is its cellular function?

XPO6 (Exportin-6) is a member of the exportin family that functions as a nuclear export protein. It shuttles between the nucleus and cytoplasm, collaborating with Ran GTPase to recognize and bind nuclear export signals of cargo proteins . XPO6 specifically mediates the nuclear export of actin and profilin-actin complexes in somatic cells . This protein plays important roles in cellular processes including nuclear-cytoplasmic transport and is also known as RANBP20, Ran-binding protein 20, KIAA0370, Exp6 .

What types of XPO6 antibodies are currently available for research?

Several types of XPO6 antibodies are available for research applications. These include rabbit polyclonal antibodies from various suppliers such as Abnova (PAB23249) , Proteintech (11408-1-AP) , and Abcam (ab72333) . These antibodies are generally unconjugated and raised against either recombinant XPO6 proteins or synthetic peptides corresponding to human XPO6 . The antibodies are predominantly of rabbit IgG isotype and are available in polyclonal form, optimized for various experimental applications.

What is the molecular weight of XPO6 protein?

The calculated molecular weight of XPO6 protein is 129 kDa, corresponding to 1125 amino acids . In western blot applications, the observed molecular weight is approximately 128 kDa . This slight difference between calculated and observed molecular weights is within normal experimental variation range for large proteins and may reflect post-translational modifications or structural properties affecting electrophoretic mobility.

What species reactivity is available for XPO6 antibodies?

Available XPO6 antibodies demonstrate different species reactivity profiles. The Abnova antibody (PAB23249) shows reactivity with human samples . The Proteintech antibody (11408-1-AP) has tested reactivity with human, mouse, and rat samples . Additionally, published literature has cited reactivity with Drosophila samples for some XPO6 antibodies . When selecting an XPO6 antibody for your research, it is important to verify the specific species reactivity required for your experimental model system.

What are the recommended applications for XPO6 antibodies?

XPO6 antibodies are suitable for multiple applications, including:

• Western Blot (WB)

• Immunohistochemistry (IHC)

• Immunofluorescence (IF)

• Immunoprecipitation (IP)

• Enzyme-Linked Immunosorbent Assay (ELISA)

Published literature has validated these applications, with multiple studies utilizing XPO6 antibodies for Western blot, immunofluorescence, and knockdown/knockout validation experiments .

What are the optimal dilutions for different applications of XPO6 antibodies?

Optimal dilutions vary by application and specific antibody. Based on available data:

It is recommended that each antibody should be titrated in your specific testing system to obtain optimal results, as performance can be sample-dependent .

How can I validate the specificity of an XPO6 antibody?

To validate the specificity of XPO6 antibodies, consider these methodological approaches:

• XPO6 knockdown/knockout validation: Use siRNA, shRNA, or CRISPR-Cas9 to reduce or eliminate XPO6 expression, then confirm specificity by demonstrating reduced or absent antibody signal . Published literature has utilized siRNA sequences targeting XPO6 and stable shRNA cell lines for this purpose .

• Immunoprecipitation followed by mass spectrometry: Perform IP with the XPO6 antibody and analyze the precipitated proteins by mass spectrometry to confirm XPO6 enrichment.

• Western blot validation: Check for a single band at the expected molecular weight (approximately 128-129 kDa) .

• Positive and negative tissue controls: Use tissues known to express different levels of XPO6, such as prostate cancer tissues (high expression) versus normal prostate tissues (lower expression) .

What positive controls should I use when working with XPO6 antibodies?

Based on validated experimental data, recommended positive controls include:

• For Western blot and IP: HeLa cell lysates, which have been validated to express detectable levels of XPO6

• For IHC: Human tissues including heart, kidney, ovary, placenta, and skin tissues have shown positive IHC detection with XPO6 antibodies

• For cancer research: Prostate cancer tissues show higher XPO6 expression compared to normal prostatic tissues and could serve as differential expression controls

How should I design experiments to study XPO6 function in nuclear export?

When designing experiments to study XPO6's nuclear export function:

• Begin with subcellular fractionation experiments to isolate nuclear and cytoplasmic fractions, followed by Western blot analysis to detect XPO6 and its cargo proteins (particularly actin and profilin-actin complexes) .

• Implement live-cell imaging with fluorescently tagged XPO6 and potential cargo proteins to track nuclear-cytoplasmic shuttling in real-time.

• Use immunofluorescence to co-localize XPO6 with its binding partners, such as Ran GTPase and actin .

• Employ gain and loss of function approaches:

  • Overexpress XPO6 using expression vectors

  • Knockdown XPO6 using validated siRNA or shRNA sequences

  • Assess the impact on nuclear export of known cargo proteins

• Consider the Ran GTPase cycle when designing experiments, as XPO6 functions in collaboration with Ran GTPase .

What are the best methods for silencing XPO6 expression in cell culture?

Several effective methods for silencing XPO6 have been validated in research:

• Transient siRNA transfection:

  • Use Lipofectamine 2000 reagent following manufacturer's instructions

  • Published siRNA sequences targeting XPO6 have been validated in previous studies

• Stable shRNA knockdown:

  • Lentiviral particles equipped with shRNAs targeting XPO6

  • Example sequence: shXPO6: GUGCCUUUCACUGAGCAAATT

  • Select stable cell lines using puromycin (2 μg/ml) for approximately 7 days

• Validate knockdown efficiency by:

  • Western blot analysis of XPO6 protein levels

  • qRT-PCR to assess XPO6 mRNA levels

These approaches have been successfully employed in prostate cancer cell lines to study XPO6 function in tumor progression and drug resistance .

How can I detect XPO6 localization in cells?

To detect XPO6 localization:

• Immunofluorescence microscopy:

  • Use XPO6 antibodies at recommended dilutions (1-4 μg/ml for Abnova antibody)

  • Co-stain with nuclear markers (DAPI) and cytoskeletal markers (actin)

  • Consider confocal microscopy for more precise localization

• Subcellular fractionation:

  • Separate nuclear and cytoplasmic fractions

  • Perform Western blot using XPO6 antibodies

  • Include appropriate fractionation controls (nuclear: lamin; cytoplasmic: GAPDH)

• Immunohistochemistry:

  • Use XPO6 antibodies at recommended dilutions (1:50-1:500 for Proteintech antibody)

  • For optimal antigen retrieval, use TE buffer pH 9.0 or citrate buffer pH 6.0

What model systems are suitable for studying XPO6 function?

Based on published research and antibody validation data, the following model systems are suitable:

• Cell lines:

  • Human: HeLa cells have been validated for XPO6 expression and antibody reactivity

  • Prostate cancer cell lines have been used to study XPO6's role in cancer progression

• Tissue models:

  • Human tissues: heart, kidney, ovary, placenta, and skin tissues have been validated for XPO6 detection

  • Prostate cancer tissues show differential expression of XPO6 compared to normal tissues

• Animal models:

  • XPO6 antibodies show reactivity with mouse and rat samples

  • Mouse xenograft models have been used to study XPO6's role in tumor progression in vivo

What is known about XPO6's role in cancer progression?

XPO6 has been implicated in cancer progression through several mechanisms:

• In prostate cancer:

  • XPO6 is upregulated in PCa tissues compared to normal tissues

  • Higher expression correlates with advanced pathological grades, including higher Gleason scores and N classification

  • XPO6 promotes tumor development in vitro and in vivo

  • Logistic regression analysis shows that higher XPO6 expression is associated with poor prognostic characteristics

• In breast cancer:

  • Reducing XPO6 levels drives anti-cancer effects via accumulating profilin-1

• In non-small-cell lung cancer:

  • XPO6 promotes cancer progression by influencing necrotic cell death

These findings suggest XPO6 could function as an oncogene across multiple cancer types, making it a potential prognostic marker and therapeutic target .

How does XPO6 contribute to chemotherapy resistance in cancer?

Research has demonstrated that XPO6 contributes to chemotherapy resistance, particularly to docetaxel (DTX) in prostate cancer:

• XPO6 knockdown enhances sensitivity to docetaxel treatment in prostate cancer cells

• Mechanistically, XPO6 regulates the Hippo pathway through:

  • Promoting YAP1 protein expression

  • Facilitating YAP1 nuclear translocation

  • These actions contribute to docetaxel resistance

• Blocking the Hippo pathway with YAP1 inhibitors reverses XPO6-mediated chemotherapeutic resistance

This suggests that targeting XPO6 could be a strategy to overcome docetaxel resistance in prostate cancer treatment .

What mechanisms underlie XPO6's effects on the Hippo pathway?

The relationship between XPO6 and the Hippo signaling pathway has been elucidated:

• XPO6 positively regulates YAP1 (Yes-associated protein 1), a key effector of the Hippo pathway

• Mechanistically, XPO6:

  • Promotes YAP1 protein expression

  • Facilitates YAP1 nuclear translocation

• When the Hippo pathway is blocked with YAP1 inhibitors, the effects of XPO6 on biological functions are lost

• This regulatory mechanism appears to be a key mechanism by which XPO6 promotes prostate cancer progression and chemotherapeutic resistance

Further research is needed to fully understand the molecular interactions between XPO6 and Hippo pathway components.

Why might I observe discrepancies in XPO6 detection between different antibodies?

Discrepancies in XPO6 detection between different antibodies may arise from several factors:

• Different epitopes:

  • Antibodies raised against different regions of XPO6 may have varying accessibility to epitopes

  • The Abnova antibody is raised against recombinant XPO6

  • The Proteintech antibody uses XPO6 fusion protein Ag1898 as immunogen

  • The Abcam antibody utilizes a synthetic peptide within human XPO6

• Post-translational modifications:

  • If the epitope region contains modifications, antibody binding may be affected

  • Different cell/tissue types may have different patterns of XPO6 modification

• Experimental conditions:

  • Each antibody has optimal buffer conditions (the Proteintech antibody is stored in PBS with 0.02% sodium azide and 50% glycerol pH 7.3)

  • Antigen retrieval methods vary (TE buffer pH 9.0 or citrate buffer pH 6.0 for IHC)

To address these discrepancies, validate multiple antibodies in your specific experimental system and include appropriate controls.

How do I optimize immunoprecipitation protocols for XPO6?

For optimal XPO6 immunoprecipitation:

• Antibody selection and concentration:

  • For the Proteintech antibody (11408-1-AP), use 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

  • For the Abcam antibody (ab72333), use 3 μg/mg whole cell lysate

• Sample preparation:

  • HeLa cells have been validated for successful IP of XPO6

  • Use fresh lysates whenever possible

  • Include protease and phosphatase inhibitors

• Protocol optimization:

  • For western blot detection after IP, the Abcam antibody has been validated at 1 μg/ml

  • Load approximately 20% of IP sample for western blot analysis

  • Use appropriate negative controls (control IgG)

• Detection:

  • Chemiluminescence with an exposure time of approximately 30 seconds has been validated

  • Expected band size is 128-129 kDa

How can I study the interaction between XPO6 and the Ran GTPase cycle?

To study the interaction between XPO6 and the Ran GTPase cycle:

• Co-immunoprecipitation experiments:

  • Immunoprecipitate XPO6 and probe for Ran GTPase, or vice versa

  • Include GTP-γS or GDP in lysates to stabilize specific nucleotide-bound states

• GST pull-down assays:

  • Use recombinant GST-tagged XPO6 to pull down Ran from cell lysates

  • Compare binding with RanGTP versus RanGDP forms

• Functional studies:

  • Employ dominant-negative Ran mutants (T24N, GDP-bound; Q69L, GTP-bound)

  • Assess their impact on XPO6-mediated nuclear export

• Visualization approaches:

  • Use proximity ligation assays (PLA) to visualize XPO6-Ran interactions in situ

  • Perform FRET experiments with fluorescently tagged XPO6 and Ran

These approaches will help elucidate how XPO6 collaborates with Ran GTPase to recognize and bind nuclear export signals of its cargo proteins .

How can XPO6 be targeted therapeutically in cancer research?

Based on emerging research, several approaches for targeting XPO6 in cancer research are promising:

• Genetic silencing approaches:

  • siRNA and shRNA targeting XPO6 have shown efficacy in reducing cancer cell proliferation and increasing chemosensitivity

  • Validated sequences for XPO6 silencing are available in the literature

• Combination therapies:

  • Combining XPO6 inhibition with docetaxel treatment enhances chemotherapeutic effects in prostate cancer models

• Indirect targeting via downstream pathways:

  • Targeting the Hippo pathway with YAP1 inhibitors may counteract XPO6-mediated effects

  • This approach focuses on blocking the downstream mechanisms of XPO6 action

• Biomarker development:

  • XPO6 expression levels could serve as a prognostic marker for cancer progression and potential chemotherapy resistance

  • This may help identify patients who would benefit from targeted therapies