TBL1XR1 Monoclonal Antibody

What is TBL1XR1 and which cellular compartments should I expect to detect it in?

TBL1XR1 is an evolutionarily conserved F-box-like protein primarily involved in transcriptional regulation. It functions as an integral subunit of the NCoR (nuclear receptor corepressor) and SMRT (silencing mediator of retinoic acid and thyroid hormone receptors) complexes . While TBL1XR1 is predominantly localized in the nucleus, cytoplasmic localization has been observed in a minority of cases (approximately 5.6% or 4/72 cases in osteosarcoma tissues) . When performing immunocytochemistry or immunohistochemistry, expect strong nuclear staining with occasional cytoplasmic signal depending on cell type and physiological state. For optimal visualization, use appropriate nuclear and cytoplasmic markers as controls when assessing subcellular localization patterns.

What are the recommended applications for TBL1XR1 monoclonal antibodies?

TBL1XR1 monoclonal antibodies have been validated for multiple applications, with varying optimal dilutions:

For ChIP applications specifically, optimal results are achieved using 10 μl of antibody and 10 μg of chromatin (approximately 4 × 10^6 cells) per immunoprecipitation . When performing Western blot, the typical observed molecular weight ranges from 50-65 kDa, with the calculated molecular weight being 56 kDa .

How should I select the appropriate TBL1XR1 monoclonal antibody clone for my experiments?

Selection of the appropriate TBL1XR1 antibody clone depends on several research parameters:

• Target region specificity: Different clones target distinct epitopes. For example, clone OTI2A8 recognizes the full-length TBL1XR1 , while other antibodies target specific amino acid regions (e.g., AA 81-178) .

• Cross-reactivity requirements: Consider whether you need species cross-reactivity. Some clones show reactivity to human, mouse, and rat samples , while others are more species-restricted .

• Application compatibility: Verify validation status for your specific application. For instance, clone D4J9C is validated for WB, IP, and ChIP , while clone 4F3-A8-D9 is validated for WB, IHC-P, and ICC/IF .

• Epitope accessibility: For complex formation studies, select antibodies targeting regions outside known protein-protein interaction domains to avoid epitope masking.

Always perform validation in your experimental system using appropriate positive and negative controls, even with pre-validated antibodies.

What is the expected molecular weight pattern for TBL1XR1 in Western blotting and how should I optimize detection?

TBL1XR1 typically appears at 50-65 kDa on Western blots, with the calculated molecular weight being 56 kDa . Variations in observed molecular weight may occur due to post-translational modifications or tissue-specific isoforms.

For optimal detection in Western blotting:

• Sample preparation: Use RIPA buffer with protease inhibitors for extraction from nuclear and cytoplasmic fractions.

• Loading controls: GAPDH is commonly used as shown in multiple studies .

• Dilution optimization: Begin with manufacturer-recommended dilutions (typically 1:500-1:2000) and adjust as needed .

• Detection conditions: For enhanced sensitivity, overnight primary antibody incubation at 4°C is recommended.

• Validation approach: Confirm specificity using knockdown or knockout controls as demonstrated in TBL1XR1 siRNA experiments in H1703 cells .

When analyzing TBL1XR1 expression across different cell lines, significant variations have been observed. For instance, osteosarcoma cell lines (MG63, U2-OS, 143B, HOS) show elevated TBL1XR1 levels compared to osteoblast cells (hFOB) at both protein and mRNA levels, with U2-OS and 143B exhibiting the highest expression .

What validation controls should I include when using TBL1XR1 antibodies in expression studies?

Rigorous validation is essential for reliable TBL1XR1 expression analysis:

• Positive tissue controls: Mouse and rat brain tissues, and MCF-7 cells have been validated as positive controls for TBL1XR1 expression .

• Genetic manipulation controls:

  • For overexpression: TBL1XR1-expressing vector systems such as those used in SK-MES-1 cells

  • For knockdown: siRNA-mediated TBL1XR1 silencing as demonstrated in H1703 cells

• Antibody controls:

  • Isotype control (matching the host species and isotype of your primary antibody)

  • Secondary antibody-only control to assess non-specific binding

• Subcellular localization controls:

  • Nuclear marker (e.g., Histone H3) when examining nuclear localization

  • Cytoplasmic marker when examining potential cytoplasmic localization

A complete validation panel includes both technical controls (antibody specificity) and biological controls (known expression patterns) to ensure reproducible results.

How should I optimize immunohistochemistry protocols for TBL1XR1 detection in tissue samples?

For optimal immunohistochemical detection of TBL1XR1:

• Antigen retrieval: Heat-mediated antigen retrieval with Tris/EDTA buffer at pH 9.0 has been successfully employed prior to staining protocols . This step is crucial as inadequate epitope exposure is a common cause of false-negative results.

• Section preparation: Use 4-5 μm thick formalin-fixed, paraffin-embedded (FFPE) tissue sections mounted on positively charged slides.

• Blocking parameters: Block with 5-10% normal serum from the same species as the secondary antibody for 1 hour at room temperature to minimize background.

• Antibody dilution: Begin testing with a 1:100 dilution for IHC-P applications , adjusting based on signal intensity and specificity.

• Visualization system: HRP Polymer for Rabbit IgG has shown excellent results with TBL1XR1 rabbit monoclonal antibodies .

• Counterstaining: Light hematoxylin counterstaining allows clear visualization of nuclear TBL1XR1 expression without obscuring specific staining.

• Expression interpretation: In osteosarcoma tissues, high TBL1XR1 expression has been observed in 74.5% (53/72) of cases, predominantly in the nucleus, with only 5.6% (4/72) showing cytoplasmic staining .

How can I effectively use TBL1XR1 antibodies to study protein-protein interactions within the NCoR/SMRT complex?

To study TBL1XR1's interactions within the NCoR/SMRT complex:

• Co-immunoprecipitation (Co-IP):

  • Use TBL1XR1 antibody at 1:100 dilution for immunoprecipitation

  • Validate with reverse IP using antibodies against known interactors (NCOR1, HDAC3)

  • Include negative controls (IgG from the same species)

  • Western blot with antibodies against interacting partners such as NCOR1 and HDAC3

• Proximity Ligation Assay (PLA):

  • Combine TBL1XR1 antibody with antibodies against potential interactors

  • Visualize protein interactions at endogenous levels with subcellular resolution

  • Quantify interaction events per cell to assess interaction frequency

• ChIP-reChIP:

  • First ChIP with TBL1XR1 antibody (1:50 dilution)

  • Second ChIP with antibodies against corepressor complex components

  • Analyze co-occupancy at specific genomic loci

Interpreting complex formation data requires consideration of regulatory stimulus effects. For example, TBL1XR1 knockout in mice affects NCOR complex stability and disrupts MAPK cascade regulation , suggesting stimulus-dependent complex dynamics.

What experimental approaches should I use to investigate TBL1XR1's role in cancer progression?

To investigate TBL1XR1's oncogenic functions:

• Expression correlation with clinical outcomes:

  • High TBL1XR1 expression correlates with poor prognosis in several cancers including osteosarcoma

  • Multivariate cox regression analysis identified TBL1XR1 as an independent prognostic factor (hazard ratio: 0.366; 95% CI: 0.349-0.460; p=0.021)

  Variable	Hazard ratio	95% confidence interval	P value
  Tumor size (≥5cm vs <5cm)	1.780	1.238-1.872	0.015
  Metastasis (present vs absent)	1.427	1.344-2.322	0.013
  Enneking staging (IA+IIA vs IIB+IIIA)	1.322	1.658-2.763	0.001
  TBL1XR1 expression (high vs low)	0.366	0.349-0.460	0.021

• Functional assays following genetic manipulation:

  • Proliferation: CCK-8 assay reveals TBL1XR1 knockdown significantly suppresses proliferation in osteosarcoma and lung SCC cells

  • Migration: Scratch wound healing and transwell assays demonstrate TBL1XR1 downregulation inhibits migration

  • Invasion: Matrigel-coated transwell assays show reduced invasion capability with TBL1XR1 knockdown

  • Epithelial-mesenchymal transition (EMT): TBL1XR1 modulates EMT marker expression (E-cadherin, ZEB1, SNAI1) through the TGF-β/Smad pathway

• Mechanistic studies:

  • miRNA regulation: TBL1XR1 is a direct target of miR-186-5p in osteosarcoma cells

  • Signaling pathway analysis: TBL1XR1 activates TGF-β/Smad signaling by increasing p-Smad2/3, Smad2, and Smad3 levels

• In vivo models:

  • Xenograft models with TBL1XR1-manipulated cancer cells

  • Analysis of tumor growth, metastasis, and survival outcomes

For comprehensive analysis, combine multiple approaches to establish causality between TBL1XR1 expression and cancer hallmarks.

How can I effectively use TBL1XR1 antibodies in chromatin immunoprecipitation (ChIP) experiments?

For optimal TBL1XR1 ChIP experiments:

• Protocol optimization:

  • Use 10 μl of TBL1XR1 antibody and 10 μg of chromatin (approximately 4 × 10^6 cells) per IP

  • Validated antibodies like TBL1XR1/TBLR1 (D4J9C) Rabbit mAb have been specifically tested for ChIP applications

  • Employ enzymatic ChIP kits for reproducible chromatin fragmentation

• Controls:

  • Positive control: ChIP with histone H3 antibody

  • Negative control: ChIP with non-specific IgG

  • Input control: Sonicated chromatin without immunoprecipitation

• Target loci selection:

  • Design primers for known NCoR/SMRT complex-regulated genes

  • Include both positive (actively regulated) and negative (not regulated) genomic regions

• Data analysis:

  • Normalize ChIP-qPCR data to input and IgG control

  • Compare enrichment across multiple genomic regions

  • Validate findings with orthogonal methods (e.g., reporter assays)

• Advanced applications:

  • ChIP-seq for genome-wide binding profile

  • ChIP-reChIP to assess co-occupancy with other transcriptional regulators

  • Integration with transcriptomic data to correlate binding with gene expression

The nuclear localization of TBL1XR1 is critical for its function in transcriptional regulation, and ChIP experiments can reveal how TBL1XR1 contributes to gene expression programs in normal development and disease states.

How does TBL1XR1 expression and function differ across cancer types and how should antibody-based detection be optimized?

TBL1XR1 expression varies significantly across cancer types, requiring tailored detection approaches:

For comprehensive cancer studies, combine TBL1XR1 expression analysis with functional assays examining relevant downstream pathways specific to each cancer type.

What experimental design is most effective for studying TBL1XR1's role in neurodevelopmental disorders?

For investigating TBL1XR1's function in neurodevelopmental contexts:

• Mouse model approaches:

  • Tbl1xr1 knockout mice exhibit behavioral and neuronal abnormalities

  • Behavioral assessment: Rotarod exercise, beam walking, and catwalk parameters reveal motor coordination impairments in knockout mice

  • Brain development analysis: Compare brain-to-body weight ratios between wild-type and knockout animals

• Cellular models:

  • Neural progenitor proliferation/differentiation assays

  • TBL1XR1 knockout in neural stem cells (NSCs) results in decreased proliferation and increased differentiation

  • Complementation experiments with wild-type vs. mutant TBL1XR1:

    • F10L (schizophrenia)

    • G70N (West syndrome-like)

    • L282P (ASD)

    • Y446C (Pierpont syndrome)

• Molecular pathway analysis:

  • MAPK cascade regulation: TBL1XR1 deficiency disrupts this pathway in neural development

  • NCOR complex stability: Western blot analysis of NCOR1 and HDAC3 levels in TBL1XR1-deficient vs. control samples

  • Wnt signaling assessment: Examine β-catenin levels and phosphorylation status

• Antibody application strategy:

  • Brain tissue immunohistochemistry: Assess TBL1XR1 expression patterns across developmental stages

  • Co-localization studies with neural markers

  • Subcellular fractionation followed by Western blot to assess compartmentalization

These approaches can elucidate how TBL1XR1 mutations or deficiency contributes to neurodevelopmental disorders with distinct clinical presentations.

How should I address inconsistent results when using TBL1XR1 monoclonal antibodies across different experimental systems?

Inconsistent results with TBL1XR1 antibodies may stem from several factors:

• Epitope accessibility issues:

  • TBL1XR1 forms complexes with multiple proteins that may mask epitopes

  • Solution: Test multiple antibodies targeting different regions (e.g., N-terminal vs. C-terminal epitopes)

  • Some antibodies recognize specific domains: AA 81-178 , full-length protein , or combinations of TBL1XR1+TBL1X+TBL1Y

• Post-translational modifications:

  • Phosphorylation or ubiquitination may affect epitope recognition

  • Solution: Use phosphatase treatment to determine if modifications affect detection

• Expression level variations:

  • TBL1XR1 expression varies significantly across cell lines and tissues

  • For instance, osteosarcoma studies show marked differences between cell lines, with U2-OS and 143B exhibiting highest expression

  • Solution: Include positive controls with known expression levels in each experiment

• Cross-reactivity considerations:

  • Some antibodies cross-react with related proteins (TBL1X, TBL1Y)

  • Solution: Use antibodies specific to TBL1XR1 when discrimination is critical, or use those that detect all family members when broader detection is desired

• Technical validation approach:

  • Perform side-by-side comparison of multiple antibody clones

  • Validate with genetic approaches (siRNA, CRISPR knockout)

  • Test antibody performance in multiple applications rather than relying on a single technique

When publishing results, clearly document the specific antibody clone, catalog number, and experimental conditions to facilitate reproducibility.

What are the critical factors for accurate quantification of TBL1XR1 levels in comparative studies?

For accurate TBL1XR1 quantification:

• Sample preparation standardization:

  • Consistent cell lysis buffers and protein extraction protocols

  • Cellular fractionation is recommended as TBL1XR1 is predominantly nuclear with occasional cytoplasmic localization

  • Equal protein loading verified by total protein staining methods

• Western blot optimization:

  • Standard curve with recombinant TBL1XR1 for absolute quantification

  • Linear dynamic range determination for your detection system

  • Multiple technical and biological replicates (minimum n=3)

• Normalization strategy:

  • Multiple housekeeping controls (e.g., GAPDH, β-actin)

  • Total protein normalization methods for more accurate comparisons

  • Consider subcellular fraction-specific loading controls (e.g., Histone H3 for nuclear fraction)

• Image acquisition parameters:

  • Avoid saturation during digital image capture

  • Consistent exposure settings across compared samples

  • Background subtraction methods applied uniformly

• Statistical analysis:

  • Appropriate statistical tests for your experimental design

  • Report both biological and technical variation

  • Consider power analysis to determine appropriate sample size

When comparing TBL1XR1 expression across different conditions or cell types, it's important to note that even modest differences may have functional significance, as evidenced by the impact of TBL1XR1 knockdown on cell proliferation, migration, and invasion in multiple cancer models .

How can I distinguish between direct and indirect effects when studying TBL1XR1's functional role using antibody-based techniques?

Distinguishing direct versus indirect TBL1XR1 effects requires a multi-faceted approach:

• Proximity-based interaction studies:

  • Proximity ligation assay (PLA) to visualize TBL1XR1 interactions with suspected direct partners

  • FRET/BRET assays for real-time interaction dynamics

  • Co-immunoprecipitation with reciprocal validation (pull down with TBL1XR1 antibody and partner antibody)

• Temporal resolution experiments:

  • Time-course studies following TBL1XR1 manipulation

  • Pulse-chase approaches to track primary versus secondary effects

  • Rapid inducible systems (e.g., auxin-inducible degron tagging of TBL1XR1)

• Domain-specific mutations:

  • Structure-function analysis with TBL1XR1 mutants

  • Point mutations disrupting specific protein interactions

  • Complementation experiments with mutant versions as performed in neural progenitor studies :

    • F10L (schizophrenia)

    • G70N (West syndrome-like)

    • L282P (ASD)

    • Y446C (Pierpont syndrome)

• Pathway inhibitor combinations:

  • Combine TBL1XR1 manipulation with inhibitors of suspected downstream pathways

  • For example, TGF-β pathway inhibitors in lung SCC studies or MAPK inhibitors in neural development studies

  • Assess rescue or synergistic effects to establish pathway relationships

• Direct binding assessment:

  • ChIP-seq for genome-wide binding patterns

  • Motif analysis to determine direct DNA binding versus co-factor recruitment

  • In vitro binding assays with purified components

These approaches collectively provide stronger evidence for direct versus indirect TBL1XR1-mediated effects than any single method alone.