JTB Antibody

What is JTB protein and why is it important in research?

JTB, also known as Jumping translocation breakpoint protein, is a 16kDa protein that plays critical roles in chromatin remodeling and gene expression regulation. It functions as a member of the Jumonji family of histone demethylases involved in epigenetic regulation . JTB is required for normal cytokinesis during mitosis and plays an important role in the regulation of cell proliferation. It may be a component of the chromosomal passenger complex (CPC), which acts as a key regulator of mitosis . The protein enables protein kinase binding activity and is involved in positive regulation of protein kinase activity . Its involvement in epigenetic processes makes it a promising target for research into cancer development, progression, and treatment.

What cellular compartments does JTB localize to?

JTB demonstrates complex subcellular localization patterns that are important to understand for proper experimental design. The protein primarily localizes to multiple cellular compartments including:

• Cytoplasm

• Cell membrane (as a single-pass type I membrane protein)

• Mitochondria

• Centrosome

• Cytoskeleton

• Microtubule organizing center

• Spindle

Interestingly, research has shown that JTB normally localizes to mitochondria, but certain conditions can alter this localization. For example, in HepG2 cells, the presence of HBsAg (Hepatitis B surface antigen) decreases the translocation of JTB to mitochondria and causes cytoplasmic accumulation of JTB . This altered localization can significantly impact cellular functions, highlighting the importance of studying JTB's subcellular distribution in different experimental contexts.

What applications are JTB antibodies validated for?

JTB antibodies are validated for multiple research applications depending on the specific antibody. For example:

• The JTB Rabbit Polyclonal Antibody (CAB10427) is validated for:

  • Western blot (WB) with recommended dilution of 1:500-1:2000

  • ELISA

• The Anti-JTB antibody (ab254640) is validated for:

  • Immunohistochemistry on paraffin-embedded sections (IHC-P)

  • Western blot (WB)

  • Immunocytochemistry/Immunofluorescence (ICC/IF)

When designing experiments, it's crucial to select an antibody that has been validated for your specific application and to follow the recommended dilutions and protocols.

How should I optimize Western blot protocols for JTB detection?

For optimal Western blot detection of JTB, consider the following methodological approach:

• Sample preparation: Use appropriate lysis buffers containing protease inhibitor cocktails to prevent protein degradation. Different cell compartments may require specific extraction protocols given JTB's diverse cellular localization.

• Gel selection: Since JTB has a calculated and observed molecular weight of 16kDa, use 12-15% SDS-PAGE gels for optimal resolution of this relatively small protein .

• Antibody dilution: For the JTB Rabbit Polyclonal Antibody (CAB10427), use a dilution range of 1:500-1:2000 . Always optimize the dilution for your specific sample type.

• Controls: Include positive control samples such as MCF7, NCI-H460, A375, rat spleen, or rat thymus tissues, which are known to express JTB .

• Loading control: β-actin has been successfully used as a loading control in Western blot analyses of JTB .

• Detection system: Use an appropriate secondary antibody against rabbit IgG, as most JTB antibodies are rabbit polyclonal.

• Validation: Confirm specificity by using siRNA knockdown controls as demonstrated in previous studies .

What are effective approaches for studying JTB localization in cells?

To effectively study JTB localization in cells, researchers can employ multiple complementary approaches:

• Subcellular fractionation and Western blotting:

  • Separate cellular components (cytoplasmic, membrane, mitochondrial, and nuclear fractions)

  • Perform Western blotting with JTB antibody

  • Use compartment-specific markers (e.g., coxIV for mitochondria) as controls

• Immunofluorescence confocal microscopy:

  • Fix cells appropriately (4% paraformaldehyde is commonly used)

  • Permeabilize cells (0.1-0.5% Triton X-100)

  • Block with appropriate blocking buffer

  • Incubate with JTB antibody at optimized dilution

  • Use fluorescently-labeled secondary antibodies

  • Co-stain with organelle markers (e.g., MitoTracker for mitochondria)

  • Analyze using confocal microscopy

• Co-immunoprecipitation for interaction studies:

  • Use whole-cell lysates and antibodies against JTB and potential binding partners

  • Perform immunoprecipitation with A/G agarose beads coated with appropriate antibodies

  • Detect interacting proteins via Western blot analysis

  • Include mouse or rabbit immunoglobulin IgGs as negative controls

This multi-method approach provides robust validation of JTB localization and potential interactions within different cellular compartments.

How can I investigate JTB's role in mitotic cytokinesis?

To investigate JTB's role in mitotic cytokinesis, consider these methodological approaches:

• JTB knockdown studies:

  • Design siRNA or shRNA constructs targeting JTB (example sequence from the literature: Forward primer 5′-CACCGCGGAAGAGTGTTCTTCATACGTGTGCTGTCCGTATGGAGAGCACTCTTCTGCTTTTT-3′)

  • Create stable knockdown cell lines using appropriate selection markers (e.g., puromycin)

  • Validate knockdown efficiency via qRT-PCR and Western blot

  • Primers for qRT-PCR validation: 5′-CGGGCTAAAACTACCCCTGAG-3′ and 5′-TGAGCGGCAGCTTTTGAACT-3′

• Live-cell imaging of mitosis:

  • Use fluorescently tagged markers for chromosomes and microtubules

  • Track cells through mitosis using time-lapse microscopy

  • Quantify cytokinesis defects, timing abnormalities, and multinucleation

• Analysis of CPC components:

  • Examine interactions between JTB and known CPC components

  • Evaluate the impact of JTB knockdown on localization and activity of CPC proteins

  • Investigate AURKB activity using phospho-specific antibodies, as JTB has been shown to increase AURKB activity

• Functional rescue experiments:

  • Re-express JTB in knockdown cells to confirm phenotype specificity

  • Use JTB mutants affecting different domains to identify critical regions for function

These approaches allow for comprehensive analysis of JTB's functional role in the complex process of mitotic cytokinesis.

How does JTB interact with HBsAg and what are the functional consequences?

The interaction between JTB and HBsAg (Hepatitis B surface antigen) represents an important area of research with implications for liver cancer development. To investigate this interaction:

• Co-immunoprecipitation analysis:

  • Immunoprecipitate with anti-JTB antibody followed by Western blot with anti-HBs antibody

  • In parallel, immunoprecipitate with anti-HBs antibody followed by Western blot with anti-JTB antibody

  • Include whole-cell lysate as positive control and immunoglobulin IgG as negative control

• Subcellular localization studies:

  • Perform immunofluorescence confocal microscopy to:

    • Confirm JTB localization to mitochondria in normal conditions

    • Demonstrate co-localization of HBs with JTB

    • Show that HBs expression decreases JTB translocation to mitochondria

    • Document cytoplasmic accumulation of JTB in the presence of HBs

• Functional consequence analysis:

  • Investigate cell motility using invasion assays and wound healing assays

  • Examine apoptotic responses, particularly in response to stressors like H₂O₂

  • Analyze the expression of downstream targets like MMP-2, which is regulated by JTB and affects cell motility

  • Evaluate the phosphorylation status of p65, as JTB inhibits p65 phosphorylation

Research has shown that JTB silencing significantly increases cell motility, which is further enhanced by HBs expression. Additionally, the JTB-HBs interaction appears to affect apoptotic pathways, with JTB knockdown in HBs-expressing cells showing enhanced anti-apoptotic activity compared to control cells .

What approaches can be used to study JTB's role in cancer progression?

To investigate JTB's potential role as a tumor suppressor and its involvement in cancer progression:

• Expression analysis in cancer tissues:

  • Analyze JTB expression levels in tumor vs. normal tissues using immunohistochemistry

  • Correlate expression with clinical parameters (stage, grade, patient survival)

  • Examine expression patterns in different cancer cell lines (e.g., HepG2 and Huh7 show high JTB expression, while SGC7901 and AGS show lower expression compared to normal cells)

• Functional studies in cancer models:

  • Create stable JTB knockdown or overexpression cell lines

  • Assess:

    • Proliferation rates

    • Colony formation ability

    • Migration and invasion capabilities

    • Resistance to apoptosis

    • In vivo tumor growth in xenograft models

• Molecular pathway analysis:

  • Investigate JTB's effect on:

    • NF-κB signaling (particularly p65 phosphorylation)

    • MMP-2 expression and activity

    • Apoptotic pathway components (Bcl-XL, caspase-9, cytochrome C, PARP)

    • Mitochondrial function (membrane potential, morphology)

• Translocation studies:

  • Examine chromosomal rearrangements involving the JTB gene

  • Analyze the consequences of these rearrangements on JTB expression and function

  • Investigate whether JTB jumping translocations correlate with tumor differentiation and staging

These approaches provide a comprehensive framework for understanding JTB's complex role in cancer biology and potentially identifying new therapeutic targets.

What are common challenges when working with JTB antibodies and how can they be addressed?

Researchers may encounter several challenges when working with JTB antibodies:

• Specificity concerns:

  • Always validate antibody specificity using positive controls (MCF7, NCI-H460, A375, rat spleen, rat thymus)

  • Include JTB knockdown controls to confirm band identity

  • Consider using multiple antibodies targeting different epitopes of JTB

• Detection of low abundance:

  • Optimize protein extraction protocols based on JTB's multiple subcellular localizations

  • Consider using enrichment techniques like subcellular fractionation

  • Increase protein loading amounts while maintaining good gel resolution

  • Use more sensitive detection systems (e.g., enhanced chemiluminescence)

• Cross-reactivity issues:

  • Use appropriate blocking reagents to minimize background

  • Optimize antibody dilutions (1:500-1:2000 for Western blot applications)

  • Include additional washing steps to reduce non-specific binding

• Variability between experiments:

  • Standardize lysate preparation protocols

  • Use consistent positive controls across experiments

  • Quantify expression levels relative to appropriate housekeeping proteins

  • Document experimental conditions thoroughly

How should I approach contradictory results in JTB localization or function studies?

When encountering contradictory results in JTB studies, consider these methodological approaches:

• Examine experimental conditions:

  • Cell type differences: JTB expression and function may vary between cell types (e.g., different expression levels observed in liver cancer vs. gastric cancer cell lines)

  • Growth conditions: Confluence, serum levels, and stress conditions may affect JTB localization

  • Disease context: HBsAg presence significantly alters JTB localization in liver cells

• Investigate post-translational modifications:

  • JTB function may be regulated by phosphorylation, ubiquitination, or other modifications

  • Different antibodies may have varying sensitivity to modified forms of JTB

• Consider interaction partners:

  • JTB function may depend on interaction partners present in specific cell types

  • The JTB-HBs interaction significantly affects JTB localization and function

• Employ multiple complementary techniques:

  • Combine biochemical approaches (Western blotting, co-IP) with microscopy

  • Use both fixed and live-cell imaging for localization studies

  • Validate functional studies with multiple approaches (e.g., siRNA and CRISPR)

• Repeat experiments with validated reagents:

  • Ensure antibody specificity through appropriate controls

  • Validate siRNA or shRNA efficiency at both mRNA and protein levels

  • Consider antibodies from different sources that target different epitopes

By systematically addressing these factors, researchers can resolve contradictory findings and develop a more comprehensive understanding of JTB biology.

How can JTB antibodies be used to study the relationship between JTB and mitochondrial function?

Research suggests that JTB plays a role in mitochondrial function, making this an important area for further investigation:

• Co-localization studies with mitochondrial markers:

  • Use JTB antibodies in combination with established mitochondrial markers (e.g., coxIV)

  • Perform super-resolution microscopy to precisely map JTB localization within mitochondrial substructures

  • Analyze changes in co-localization under different cellular conditions

• Mitochondrial function assays:

  • Investigate how JTB knockdown or overexpression affects:

    • Mitochondrial membrane potential

    • Mitochondrial morphology (JTB overexpression has been associated with mitochondrial swelling)

    • Oxygen consumption rate

    • ATP production

    • Reactive oxygen species generation

• Interaction with mitochondrial proteins:

  • Use co-immunoprecipitation with JTB antibodies to identify mitochondrial binding partners

  • Employ proximity labeling approaches (BioID or APEX) with JTB as the bait to identify neighboring proteins in the mitochondrial environment

  • Validate identified interactions through reciprocal co-immunoprecipitation and functional studies

• Response to mitochondrial stress:

  • Examine JTB localization and function under conditions that induce mitochondrial stress (e.g., oxidative stress, mitochondrial toxins)

  • Assess whether JTB plays a protective or detrimental role in the mitochondrial stress response

  • Investigate the relationship between JTB and apoptotic pathways that involve mitochondria

This research direction could provide valuable insights into JTB's role in mitochondrial biology and potentially reveal new therapeutic targets for diseases involving mitochondrial dysfunction.

What is the significance of JTB in epigenetic regulation and how can it be studied?

As a member of the Jumonji family of histone demethylases, JTB may play important roles in epigenetic regulation:

• Chromatin immunoprecipitation (ChIP) studies:

  • Use JTB antibodies for ChIP to identify genomic regions where JTB binds

  • Perform ChIP-seq to generate genome-wide binding profiles

  • Analyze binding sites for common sequence motifs or enrichment near specific gene classes

• Histone modification analysis:

  • Investigate how JTB knockdown or overexpression affects specific histone modifications

  • Focus on methyl marks (particularly H3K4, H3K9, H3K27, and H3K36 methylation)

  • Perform Western blot and immunofluorescence analyses with modification-specific antibodies

• Transcriptome analysis:

  • Use RNA-seq to identify genes regulated by JTB

  • Compare transcriptional changes upon JTB manipulation with changes in histone modifications

  • Validate key target genes through qRT-PCR and reporter assays

• Protein complex identification:

  • Use JTB antibodies for immunoprecipitation followed by mass spectrometry

  • Identify interactions with known chromatin modifiers or transcriptional regulators

  • Validate key interactions and assess their functional significance

• Correlation with disease states:

  • Analyze JTB expression and localization in conditions with known epigenetic dysregulation

  • Investigate whether JTB jumping translocations are associated with altered epigenetic profiles

  • Examine potential epigenetic mechanisms underlying JTB's reported tumor suppressor function

This research direction could significantly advance our understanding of JTB's role in epigenetic regulation and potentially reveal new therapeutic approaches for diseases with epigenetic components.