TXNL4B Antibody

Which cancer types demonstrate significant TXNL4B overexpression?

TXNL4B expression analysis across multiple cancer types reveals significant upregulation in several malignancies. According to RNA-sequencing data from the GEPIA (Gene Expression Profiling Interactive Analysis) database, TXNL4B is overexpressed in:

• COAD (colon adenocarcinoma)

• ESCA (esophageal carcinoma)

• LUSC (lung squamous cell carcinoma)

• PAAD (adenocarcinoma of the pancreas)

• SARC (sarcoma)

• STAD (stomach adenocarcinoma)

What are the recommended antibody dilutions for different TXNL4B detection methods?

Based on published research protocols, the following antibody dilutions are recommended when working with anti-TXNL4B antibodies:

These dilutions serve as starting points and may require optimization based on specific experimental conditions and antibody lots .

How should researchers design TXNL4B knockdown experiments to study radioresistance?

When designing TXNL4B knockdown experiments to study radioresistance, researchers should consider the following methodological approach:

• Selection of appropriate knockdown strategy: Use shRNA for stable knockdown or siRNA for transient knockdown. The validated shRNA sequence targeting TXNL4B is 5′-GCTTCCTACTGCCCAAGCT-3′, while a non-targeting control sequence is 5′-AATAAGGGCAATAACCCAG-3′ .

• Vector selection: For stable knockdown, clone shRNAs into a lentiviral vector system such as pGreen puro shRNA vector (SystemBio) .

• Verification of knockdown efficiency: Confirm TXNL4B knockdown at both protein (Western blot) and mRNA (qRT-PCR) levels before proceeding with radiation experiments.

• Radiation parameters: Administer ionizing radiation in controlled doses (e.g., 4-6 Gy) and establish appropriate time points for analysis (2h, 4h, 6h, 8h, and 24h post-radiation) .

• Functional assessments: Analyze multiple parameters including:

  • Cell cycle distribution using flow cytometry

  • Apoptosis rates

  • DNA damage repair through γH2AX foci formation

  • Expression of DNA repair proteins (BRCA1, 53BP1)

  • Cell proliferation and survival rates

  • EMT markers (E-cadherin, N-cadherin, Vimentin)

• In vivo validation: Consider xenograft models to confirm in vitro findings, with tumor volume measurements following combined TXNL4B knockdown and radiation treatment .

What controls are essential when studying TXNL4B-mediated radioresistance?

When investigating TXNL4B-mediated radioresistance, several critical controls must be incorporated:

• Non-targeting shRNA/siRNA control: Essential for distinguishing specific TXNL4B knockdown effects from non-specific RNA interference effects .

• Non-irradiated controls: Both knockdown and control cells without radiation exposure are necessary to establish baseline effects of TXNL4B modulation independent of radiation .

• Time course controls: Analysis at multiple time points post-radiation (2h, 4h, 6h, 8h, 24h) to capture dynamic changes in cellular responses .

• Rescue experiments: Re-expression of TXNL4B in knockdown cells to confirm observed phenotypes are specifically due to TXNL4B depletion .

• Multiple cell lines: Validation in additional lung cancer cell lines beyond A549 to ensure findings are not cell line-specific .

• Normal tissue controls: Comparison with normal lung cell lines to establish cancer-specific effects .

• Antibody validation controls: When using TXNL4B antibodies, include overexpression and knockdown samples to verify antibody specificity .

What are the optimal protocols for detecting TXNL4B-PRP3 interactions?

To effectively investigate the interaction between TXNL4B and PRP3, researchers should employ the following optimized immunoprecipitation (IP) protocol:

• Protein extraction: Extract total cellular proteins from cells exposed to experimental conditions (e.g., pre- and post-radiation treatment) .

• Pre-clearing: Incubate 500 μg of protein extract with Protein G PLUS-Agarose for 30 minutes at 4°C to reduce non-specific binding.

• Antibody incubation: Incubate pre-cleared lysates with 1 μg of anti-TXNL4B or anti-PRP3 antibody at 4°C for 1 hour .

• Immunoprecipitation: Add 20 μL Protein G PLUS-Agarose and incubate overnight at 4°C with gentle rotation .

• Washing: After centrifugation at 1000g, carefully wash the immunoprecipitates 3-4 times with cold PBS containing protease inhibitors .

• Elution and analysis: Elute bound proteins by boiling in SDS sample buffer and analyze by Western blotting for interacting partners .

• Reciprocal IP: Confirm interactions by performing reciprocal IPs (using anti-PRP3 for immunoprecipitation and anti-TXNL4B for detection, and vice versa) .

• Co-localization studies: Complement IP results with immunofluorescence co-localization experiments to visualize TXNL4B-PRP3 interactions in cellular compartments before and after radiation .

How can researchers effectively study TXNL4B nuclear translocation after radiation?

Studying TXNL4B nuclear translocation following radiation requires a multifaceted approach:

• Subcellular fractionation: Separate nuclear and cytoplasmic fractions using established protocols, followed by Western blot analysis to quantify TXNL4B distribution between compartments at various time points post-radiation .

• Immunofluorescence microscopy:

  • Fix cells at defined intervals after radiation (2h, 4h, 6h, 8h, 24h)

  • Permeabilize with 0.1% Triton X-100

  • Block with 3-5% BSA

  • Incubate with anti-TXNL4B antibody (1:200-1:400 dilution)

  • Counterstain nuclei with DAPI

  • Analyze using confocal microscopy to quantify nuclear/cytoplasmic signal ratios

• Live-cell imaging: For dynamic analysis, create TXNL4B-GFP fusion constructs and monitor nuclear translocation in real-time following radiation exposure.

• Co-localization studies: Perform dual immunofluorescence for TXNL4B and PRP3 to track their co-localization dynamics after radiation treatment .

• Mutation analysis: Generate nuclear localization signal (NLS) mutants of TXNL4B to identify domains responsible for radiation-induced nuclear translocation.

What techniques are recommended for studying TXNL4B's role in alternative splicing?

To investigate TXNL4B's involvement in alternative splicing regulation, particularly regarding FANCI transcript variants, researchers should implement these methodological approaches:

How can researchers quantify changes in TXNL4B expression following radiation treatment?

To accurately quantify radiation-induced changes in TXNL4B expression, researchers should employ multiple complementary techniques:

• Western blotting:

  • Harvest cells at various time points post-radiation (4, 8, 12, 24, 48 hours)

  • Use anti-TXNL4B antibody (1:1000 dilution, Proteintech 12927-1-AP)

  • Normalize to appropriate loading controls (β-actin, GAPDH)

  • Perform densitometric analysis using software like ImageJ for quantification

• Quantitative RT-PCR:

  • Extract total RNA using standard protocols

  • Synthesize cDNA and perform qPCR with TXNL4B-specific primers

  • Normalize to stable reference genes (GAPDH, β-actin, or 18S rRNA)

  • Calculate fold changes using the 2^-ΔΔCt method

• Immunofluorescence microscopy:

  • Fix cells at defined intervals following radiation

  • Perform immunofluorescence staining with anti-TXNL4B antibody

  • Acquire images under identical exposure settings

  • Quantify fluorescence intensity using image analysis software

  • Calculate nuclear/cytoplasmic signal ratios to assess compartmental changes

• Flow cytometry:

  • Fix and permeabilize cells

  • Stain with fluorophore-conjugated anti-TXNL4B antibody

  • Analyze mean fluorescence intensity as a measure of protein expression

How should researchers validate TXNL4B antibody specificity?

Validating TXNL4B antibody specificity is critical for reliable research outcomes. A comprehensive validation approach includes:

• Knockdown/knockout controls: Test antibody against TXNL4B-depleted samples using:

  • siRNA-mediated knockdown (validated sequence: GCUUCCUACUGCCCAAGCU)

  • shRNA-mediated stable knockdown

  • CRISPR/Cas9-mediated knockout

• Overexpression controls: Test antibody against samples overexpressing TXNL4B using:

  • Lentiviral vector GV367 containing the full-length sequence of human TXNL4B

  • Tagged TXNL4B constructs (FLAG, HA, or GFP) for dual detection systems

• Peptide competition: Pre-incubate antibody with the immunizing peptide to confirm signal elimination in positive samples.

• Multiple antibodies: Validate findings using antibodies from different manufacturers or targeting different epitopes.

• Cross-reactivity assessment: Test the antibody against samples from multiple species if conducting comparative studies.

• Multiple applications: Confirm consistent results across different detection methods (Western blot, immunofluorescence, immunoprecipitation) .

• Tissue expression profile: Compare antibody detection pattern with known TXNL4B expression profiles across tissues and cell types .

What are common technical challenges when studying TXNL4B and how can they be addressed?

Researchers working with TXNL4B may encounter several technical challenges that can be addressed with the following strategies:

• Non-specific antibody binding:

  • Solution: Optimize blocking conditions (5% BSA or milk)

  • Increase washing frequency and duration

  • Pre-absorb antibody with cell lysates from knockdown samples

• Low signal-to-noise ratio in immunofluorescence:

  • Solution: Test different fixation methods (4% paraformaldehyde, methanol)

  • Optimize antibody concentration and incubation time

  • Use signal amplification systems for low abundance detection

• Inconsistent knockdown efficiency:

  • Solution: Test multiple siRNA/shRNA sequences

  • Optimize transfection conditions

  • Use the validated sequence (GCUUCCUACUGCCCAAGCU) at appropriate concentrations

• Difficulty detecting radiation-induced changes:

  • Solution: Test multiple radiation doses (2Gy, 4Gy, 6Gy)

  • Expand time course analysis (2h, 4h, 6h, 8h, 24h, 48h)

  • Consider cell type-specific differences in radiation response

• Variable results in co-immunoprecipitation:

  • Solution: Test different lysis buffers to preserve protein interactions

  • Optimize antibody-to-lysate ratios

  • Consider crosslinking to stabilize transient interactions

How does TXNL4B influence DNA damage repair pathways after radiation?

TXNL4B plays a significant role in DNA damage repair following radiation exposure through several mechanisms:

• Regulation of DNA repair proteins: TXNL4B affects the expression and function of key DNA repair proteins, including BRCA1 and 53BP1. Research shows that TXNL4B knockdown alters the radiation-induced response of these proteins, with BRCA1 expression increasing and 53BP1 decreasing compared to control cells .

• γH2AX dynamics: TXNL4B influences the formation and resolution of γH2AX foci, a classic marker of DNA double-strand breaks. In TXNL4B knockdown cells, γH2AX foci persist longer after radiation (up to 6-8 hours post-radiation), indicating delayed DNA damage repair .

• Cell cycle checkpoint regulation: TXNL4B knockdown leads to increased G2/M arrest following radiation exposure, with approximately 50% of knockdown cells remaining in G2/M phase at 24 hours post-radiation. This prolonged checkpoint activation correlates with delayed DNA repair capacity .

• Alternative splicing regulation: Through its interaction with PRP3, TXNL4B influences the alternative splicing of FANCI transcript variants (FANCI-12 and FANCI-13), which in turn affects the DNA damage response and repair pathways .

• Correlation with DNA repair markers: Analysis shows positive correlation between TXNL4B expression and both BRCA1 and γH2AX expression, further supporting its involvement in DNA repair mechanisms .

What is the relationship between TXNL4B and the spliceosome in radioresistance?

TXNL4B functions as a key regulator of splicing machinery components that contribute to radioresistance through the following mechanisms:

• Interaction with PRP3: TXNL4B directly interacts with PRP3 (RNA processing factor 3) and co-localizes in the nucleus following radiation exposure. This interaction is critical for downstream splicing events that influence radioresistance .

• Regulation of U4/U6.U5 tri-snRNP complex: TXNL4B knockdown affects the stability of the U4/U6.U5 tri-snRNP complex, particularly through modulating the interaction between PRP31 and PRP8, which are critical components of the core spliceosome .

• Alternative splicing of FANCI: Through its spliceosomal function, TXNL4B influences the alternative splicing of FANCI into two transcript variants (FANCI-12 and FANCI-13), with FANCI-12 playing a significant role in promoting radioresistance .

• PRP3 nuclear localization: TXNL4B regulates the nuclear localization of PRP3 following radiation, which is essential for the alternative splicing events that contribute to radioresistance .

• Splicing factor interactions: The inhibition of PRP3 (influenced by TXNL4B) suppresses the production of FANCI-12, thereby disrupting the interaction between PRP31 and PRP8, which ultimately affects radioresistance mechanisms .

This complex interplay between TXNL4B, splicing factors, and alternative splicing represents a novel mechanism by which cancer cells develop radioresistance, offering potential targets for improving radiotherapy efficacy .

What are promising therapeutic approaches targeting TXNL4B for enhancing radiotherapy?

Based on current understanding of TXNL4B's role in radioresistance, several therapeutic strategies merit investigation:

• Targeted TXNL4B inhibition: Development of small molecule inhibitors or peptide-based antagonists specifically targeting TXNL4B function could enhance radiosensitivity. Research shows TXNL4B knockdown significantly increases cancer cell sensitivity to radiation .

• Splicing modulation: Compounds targeting the interaction between TXNL4B and PRP3 could disrupt alternative splicing events that contribute to radioresistance, particularly the generation of the FANCI-12 variant .

• Isoform-specific targeting: Therapeutic approaches specifically targeting the FANCI-12 splice variant could enhance radiation sensitivity without affecting normal FANCI function .

• Combined checkpoint inhibition: Since TXNL4B knockdown promotes G2/M arrest, combining TXNL4B inhibition with cell cycle checkpoint inhibitors might synergistically enhance radiation effects .

• RNA-based therapeutics: Antisense oligonucleotides or siRNAs targeting TXNL4B or specific splice junctions in FANCI could provide highly specific therapeutic options .

• EMT inhibition: Given TXNL4B's association with EMT processes, combining anti-TXNL4B strategies with EMT inhibitors might prevent radioresistance development and metastatic progression .

These approaches require further validation in preclinical models before advancing to clinical investigations. Current research limitations include the need for studies across multiple cancer cell lines and confirmation in mouse xenograft models .

What experimental approaches would help clarify contradictory findings about TXNL4B function?

To address potential contradictions in TXNL4B functional studies, researchers should consider the following experimental approaches:

• Tissue-specific analysis: Comprehensive analysis of TXNL4B functions across different tissue types, as effects may be context-dependent based on tissue origin .

• Temporal dynamics investigation: Detailed time-course experiments following radiation to capture dynamic changes in TXNL4B localization, interactions, and downstream effects .

• Isoform-specific studies: Investigation of potential TXNL4B isoforms that might have distinct functions in different cellular contexts .

• Comprehensive interaction mapping: Proteome-wide interaction studies before and after radiation to identify context-specific binding partners beyond PRP3 .

• In vivo validation: Testing observed mechanisms in multiple mouse models to validate findings beyond cell culture systems .

• Single-cell analysis: Application of single-cell RNA-seq to identify cell-specific responses that might be masked in bulk analysis .

• Multi-omics integration: Combining transcriptomics, proteomics, and functional genomics to develop a comprehensive model of TXNL4B function in radioresistance .

• Radiation type comparison: Evaluating TXNL4B's role across different radiation modalities (photon, proton, carbon ion) to determine if mechanisms are radiation-type specific .