btf3l4 Antibody

What is BTF3L4 and why is it significant in current research?

BTF3L4 (Basic Transcription Factor 3-like 4) is a transcription factor protein with a molecular weight of approximately 17 kDa that plays significant roles in basic transcription processes required for diverse biological functions . It belongs to the BTF3 family of transcription factors, which are increasingly recognized for their involvement in digestive diseases . Recent proteomic analyses have identified BTF3L4 as the only outlier transcription factor significantly overexpressed in acetaminophen (APAP)-induced liver injury models in mice . BTF3L4's significance stems from its emerging role in regulating apoptosis, inflammatory responses, and mitochondrial function, particularly in liver pathology . Unlike other BTF3 family members that have been studied in various cancers, BTF3L4's specific functions in liver injury represent a novel area of investigation with potential diagnostic and therapeutic implications.

How do BTF3L4 antibodies differ in terms of species reactivity and applications?

BTF3L4 antibodies are available with varying specificities and applications, primarily showing reactivity to human and mouse samples . Commercially available antibodies are typically generated in rabbits as polyclonal antibodies that recognize endogenous levels of total BTF3L4 protein . These antibodies differ in their validated applications, with some optimized for Western blotting (WB), immunohistochemistry (IHC) on paraffin-embedded tissues, and enzyme-linked immunosorbent assays (ELISA) . The concentration of these antibodies varies by manufacturer, with some provided at approximately 1 mg/mL or 0.8 mg/mL . Species reactivity is an important consideration when selecting an antibody for research, as antibodies that cross-react with both human and mouse BTF3L4 allow for translational research between animal models and human samples. Some antibodies are also available with PE (phycoerythrin) conjugation for fluorescence-based applications .

What are the optimal storage and handling conditions for BTF3L4 antibodies?

BTF3L4 antibodies require specific storage and handling conditions to maintain their functionality and specificity. Most commercial BTF3L4 antibodies should be stored at -20°C for long-term preservation . These antibodies are typically supplied as buffered aqueous solutions, often containing glycerol (up to 40%) and preservatives like sodium azide (0.05% NaN3) . During shipping, the antibodies are generally transported on wet ice to maintain stability . For working solutions, it's advisable to prepare only the amount needed for immediate use and avoid repeated freeze-thaw cycles, which can compromise antibody quality. When handling BTF3L4 antibodies for immunohistochemistry applications, optimal dilution ranges from 1:50 to 1:100, while for ELISA applications, much higher dilutions (around 1:40000) may be required . Proper handling includes avoiding contamination and adhering to manufacturer-specified storage conditions to prevent degradation or loss of immunoreactivity over time.

What are the validated methods for detecting BTF3L4 expression in liver tissue samples?

For detecting BTF3L4 expression in liver tissues, immunohistochemistry (IHC) and immunofluorescence are well-validated methods as demonstrated in recent APAP-induced liver injury studies . When performing IHC, formalin-fixed, paraffin-embedded liver tissue sections should undergo proper antigen retrieval in buffer according to manufacturer protocols, followed by blocking endogenous peroxidase activity with 3% H₂O₂ for 15 minutes . Primary anti-BTF3L4 antibodies (such as ab128870 from Abcam) are typically incubated overnight at 4°C at dilutions of 1:50-1:100 . For visualization, fluorescence-labeled secondary antibodies (such as ab150075 or ab150113) are incubated for 1 hour at room temperature . Western blotting serves as a complementary approach for quantitative assessment of BTF3L4 protein levels, while qPCR using primers specific for BTF3L4 (forward: 5′-AGAAGGTGGTACATAGGACAGC-3′; reverse: 5′-CCGTGCCATCGTCTTTAATCAT-3′) enables mRNA expression analysis . For studying BTF3L4 in the context of apoptosis, combining BTF3L4 immunostaining with TUNEL assays using kits like the In Situ Cell Death Detection TUNEL Kit provides valuable insights into the relationship between BTF3L4 expression and cell death in liver injury models .

How should researchers design BTF3L4 knockdown and overexpression experiments?

When designing BTF3L4 knockdown or overexpression experiments to study its functional roles, researchers should consider both lentiviral and adenoviral vector systems, which have been successfully employed in recent studies . For BTF3L4 knockdown, lentiviral plasmids encoding short hairpin RNAs (shRNAs) targeting BTF3L4 should be designed with appropriate negative control shRNAs (shNC) . For overexpression studies, adenoviral vectors containing the BTF3L4 gene sequence (Ad-BTF3L4) with proper controls are recommended . Transfection of these constructs into cell lines such as AML-12 (a mouse liver cell line) can be performed using Lipofectamine 3000 following manufacturer protocols . It is crucial to validate the efficiency of knockdown or overexpression at both mRNA and protein levels using qPCR and Western blotting, respectively . For in vivo studies, careful consideration of delivery methods is essential to ensure hepatocyte-specific expression. When assessing the functional consequences of BTF3L4 modulation, researchers should incorporate comprehensive analyses of apoptosis markers, inflammatory cytokines (IL-1β, TNF-α), reactive oxygen species levels, and mitochondrial function parameters to fully understand the downstream effects of BTF3L4 altered expression .

What controls and validation steps are essential when using BTF3L4 antibodies?

When using BTF3L4 antibodies, implementing rigorous controls and validation steps is crucial for ensuring reliable and reproducible results. Positive controls should include tissues or cell lines known to express BTF3L4, such as liver tissues from APAP-treated mice where BTF3L4 is overexpressed . Negative controls should incorporate samples where BTF3L4 is knocked down using shRNA or tissues from species not recognized by the antibody . Antibody validation should include Western blotting to confirm specificity by verifying a single band at the expected molecular weight of 17 kDa . For immunohistochemistry applications, an isotype control (using matched IgG from the same species as the primary antibody) should be run in parallel to assess non-specific binding . Additionally, peptide competition assays, where the antibody is pre-incubated with the immunizing peptide before application to the sample, can confirm binding specificity. When performing double-labeling experiments, such as co-staining with albumin (a hepatocyte marker) and BTF3L4, appropriate controls for cross-reactivity between secondary antibodies are necessary . Finally, researchers should validate antibody performance in their specific experimental conditions by testing different dilutions and incubation times to optimize signal-to-noise ratios before proceeding with full experiments.

How can BTF3L4 expression be quantified in relation to the progression of APAP-induced liver injury?

Quantifying BTF3L4 expression across the temporal progression of APAP-induced liver injury requires a multifaceted approach combining protein and mRNA analyses with histological assessment. Based on established AILI mouse models, researchers should design time-course experiments with multiple timepoints (3h, 6h, 12h, 24h, and 48h post-APAP administration) to capture the dynamic changes in BTF3L4 expression . Immunohistochemistry using validated anti-BTF3L4 antibodies (dilution 1:50-1:100) on liver sections allows for visualization of spatial distribution and semi-quantitative analysis of expression levels . For precise quantification, Western blot analysis of liver tissue homogenates at each timepoint provides relative protein expression levels that can be normalized to loading controls. At the transcriptional level, qPCR with BTF3L4-specific primers enables measurement of mRNA expression changes . To correlate BTF3L4 expression with liver injury progression, researchers should simultaneously assess standard liver injury markers (ALT, AST) in serum samples and histopathological scores using H&E staining . Advanced image analysis software can be employed to quantify BTF3L4 immunostaining intensity across different zones of the liver lobule, providing insights into zone-specific expression patterns that may correlate with the characteristic centrilobular pattern of APAP-induced hepatotoxicity.

What methodologies are most effective for investigating BTF3L4's role in mitochondrial dysfunction?

Investigating BTF3L4's role in mitochondrial dysfunction requires specialized methodologies focusing on mitochondrial morphology, function, and related signaling pathways. Confocal microscopy using mitochondria-specific fluorescent dyes (MitoTracker) in combination with immunofluorescence staining for BTF3L4 enables visualization of mitochondrial morphological changes in response to BTF3L4 overexpression or knockdown in hepatocytes . Transmission electron microscopy provides higher-resolution analysis of ultrastructural mitochondrial alterations. For functional assessments, oxygen consumption rate (OCR) measurements using platforms like Seahorse XF Analyzer quantify mitochondrial respiration parameters in cells with manipulated BTF3L4 levels. Mitochondrial membrane potential can be evaluated using JC-1 or TMRE dyes, while reactive oxygen species (ROS) generation—a key consequence of BTF3L4-mediated mitochondrial dysfunction—should be quantified using fluorescent probes such as DCFDA or MitoSOX Red . Biochemical assays measuring activities of mitochondrial electron transport chain complexes provide insights into specific functional deficits. At the molecular level, researchers should assess expression of mitochondrial fission/fusion proteins (DRP1, MFN1/2, OPA1) via Western blotting to determine if BTF3L4 influences mitochondrial dynamics. Analysis of mitochondrial DNA integrity and copy number using qPCR can reveal if BTF3L4 affects mitochondrial biogenesis or DNA stability. These methodologies, when systematically applied to BTF3L4-manipulated systems, comprehensively characterize its mechanistic involvement in mitochondrial dysfunction during liver injury.

How does BTF3L4 interact with inflammatory signaling pathways in hepatocytes?

The interaction between BTF3L4 and inflammatory signaling pathways in hepatocytes involves complex molecular mechanisms that can be investigated through several complementary approaches. To understand these interactions, researchers should examine the effects of BTF3L4 overexpression or knockdown on key inflammatory pathways using Western blotting to assess activation (phosphorylation) of signaling mediators including NF-κB, JNK, and STAT3 . Quantitative PCR analysis of inflammatory cytokine and chemokine expression (IL-1β, TNF-α, MCSFR) using validated primers (as specified in the literature: Mouse IL-1β forward, 5′-GAAATGCCACCTTTTGACAGTG-3′; Mouse IL-1β reverse, 5′-TGGATGCTCTCATCAGGACAG-3′; Mouse TNF-α forward, 5′-GATCGGTCCCCAAAGGGATG-3′; Mouse TNF-α reverse, 5′-TTTGCTACGACGTGGGCTAC-3′) enables assessment of downstream inflammatory responses . Chromatin immunoprecipitation (ChIP) assays can determine whether BTF3L4, as a transcription factor, directly binds to promoter regions of inflammatory genes. Co-immunoprecipitation experiments help identify protein-protein interactions between BTF3L4 and components of inflammatory signaling complexes. For in vivo validation, comparative analysis of inflammatory markers in liver tissues from wild-type mice and those with hepatocyte-specific BTF3L4 overexpression following APAP treatment provides physiological relevance . Additionally, fluorescence resonance energy transfer (FRET) or proximity ligation assays (PLA) can be utilized to visualize direct interactions between BTF3L4 and inflammatory signaling proteins within intact cells. Inflammatory cell infiltration in liver tissues, assessed by immunostaining for macrophage/neutrophil markers, should be correlated with hepatocyte BTF3L4 expression levels to establish connections between BTF3L4-mediated signaling and the inflammatory microenvironment.

What are common technical challenges when using BTF3L4 antibodies in liver tissue analysis?

When using BTF3L4 antibodies for liver tissue analysis, researchers commonly encounter several technical challenges that require specific troubleshooting approaches. Background staining issues in immunohistochemistry applications can be addressed by optimizing blocking conditions (using 3-5% BSA or serum from the secondary antibody host species) and ensuring proper endogenous peroxidase blocking with 3% H₂O₂ . Variability in staining intensity between samples may result from inconsistent fixation; standardizing tissue collection and fixation protocols (formalin fixation for 24 hours followed by paraffin embedding) can minimize this issue . Antigen masking is particularly problematic in liver tissues due to their high protein content; researchers should evaluate multiple antigen retrieval methods (heat-induced epitope retrieval in citrate buffer pH 6.0 vs. EDTA buffer pH 9.0) to determine optimal conditions for BTF3L4 detection . Non-specific nuclear staining can be reduced by carefully titrating primary antibody concentration (testing dilutions from 1:50 to 1:200) and including appropriate blocking steps . For dual immunofluorescence labeling with other hepatocyte markers like albumin, spectral overlap between fluorophores may confound interpretation; sequential rather than simultaneous staining and careful selection of non-overlapping fluorophores can mitigate this issue . Additionally, BTF3L4's relatively low abundance in normal liver tissue may necessitate signal amplification techniques such as tyramide signal amplification or higher antibody concentrations for reliable detection in control samples compared to APAP-treated tissues where the protein is overexpressed .

How should researchers interpret conflicting BTF3L4 expression data between different experimental models?

When faced with conflicting BTF3L4 expression data between different experimental models, researchers should implement a systematic analytical approach. First, carefully evaluate the experimental conditions across models, considering differences in species (human vs. mouse), cell types (primary hepatocytes vs. cell lines), and experimental contexts (in vitro vs. in vivo) . The temporal dynamics of BTF3L4 expression following APAP treatment vary significantly (3h, 6h, 12h, 24h, and 48h timepoints show different expression patterns); therefore, inconsistencies may reflect different sampling timepoints rather than true contradictions . Methodological differences in BTF3L4 detection (protein vs. mRNA, antibody clones, detection techniques) can significantly impact results; researchers should validate findings using multiple independent techniques (Western blot, qPCR, immunohistochemistry) with appropriate controls . Zone-specific expression patterns within the liver lobule may explain discrepancies if sampling methods differ between studies; whole-tissue analysis versus zone-specific laser capture microdissection can yield different results . Genetic background variations between mouse strains or donor-to-donor variability in human samples may contribute to expression differences. When examining BTF3L4's relationship with liver injury markers, consider that correlation patterns may differ based on injury severity or progression stage . To resolve conflicts, researchers should conduct side-by-side comparisons under standardized conditions, implement rigorous statistical analysis with appropriate sample sizes, and consider potential confounding variables such as age, sex, underlying pathologies, or concurrent medications in clinical samples.

What analytical approaches best demonstrate the relationship between BTF3L4 expression and apoptotic/inflammatory markers?

To effectively demonstrate relationships between BTF3L4 expression and apoptotic/inflammatory markers, researchers should employ multiple analytical approaches that provide complementary perspectives. Correlation analysis using Pearson or Spearman methods quantitatively assesses the strength of association between BTF3L4 expression levels and markers such as cleaved caspase-3, TUNEL-positive cells, or inflammatory cytokines (IL-1β, TNF-α) across different samples . Dual immunofluorescence co-localization studies visualize spatial relationships between BTF3L4 and apoptotic/inflammatory markers at the cellular level; quantification of co-localization coefficients (Mander's or Pearson's) provides statistical rigor to these observations . For mechanistic insights, pathway analysis following BTF3L4 manipulation (overexpression/knockdown) using Western blotting for key signaling molecules (p-JNK, p-p38, cleaved PARP) establishes causal relationships rather than mere correlations . Temporal sequence analysis tracking BTF3L4 expression changes followed by apoptotic/inflammatory events across multiple timepoints (3h, 6h, 12h, 24h, 48h post-APAP) helps establish whether BTF3L4 upregulation precedes or follows these processes . Dose-response relationships between BTF3L4 expression levels and severity of apoptosis/inflammation provide further evidence of functional connections. For broader analysis, gene set enrichment approaches using transcriptomic data from BTF3L4-manipulated systems can identify enriched pathways related to apoptosis and inflammation. Finally, in vivo intervention studies where BTF3L4 is specifically inhibited during APAP-induced liver injury, followed by assessment of apoptotic/inflammatory marker changes, provide the strongest evidence for causal relationships between BTF3L4 and these pathological processes .

What novel approaches could enhance the specificity and sensitivity of BTF3L4 detection in clinical samples?

Enhancing BTF3L4 detection in clinical samples requires innovative methodological advances beyond current antibody-based techniques. Developing highly specific monoclonal antibodies against unique BTF3L4 epitopes would significantly improve detection specificity compared to existing polyclonal antibodies . Implementation of proximity ligation assays (PLA), which provide signal amplification through DNA rolling circle amplification, could substantially enhance sensitivity for detecting low-abundance BTF3L4 in normal liver tissues. RNAscope in situ hybridization technology would enable visualization of BTF3L4 mRNA with single-molecule sensitivity while maintaining contextual information within tissue architecture. For clinical application, developing multiplex immunofluorescence panels incorporating BTF3L4 alongside established liver injury markers would provide comprehensive pathological assessment from limited biopsy material. Mass spectrometry imaging (MSI) techniques could map BTF3L4 spatial distribution across tissue sections without antibody dependency. Computational approaches integrating artificial intelligence algorithms for automated image analysis would improve quantification accuracy and reduce observer bias in BTF3L4 immunostaining interpretation. Development of aptamer-based detection systems as alternatives to antibodies could offer advantages in terms of stability, reproducibility, and cost-effectiveness. For liquid biopsy applications, exploring whether BTF3L4 or its fragments are detectable in circulation during liver injury could provide minimally invasive biomarkers. Finally, CRISPR-based techniques that specifically label endogenous BTF3L4 with fluorescent tags in live cells or organoids derived from patient samples would enable dynamic monitoring of expression patterns during disease progression or therapeutic interventions.

How might BTF3L4 be exploited as a therapeutic target or biomarker in liver pathologies?

Exploiting BTF3L4 as a therapeutic target or biomarker in liver pathologies presents several promising research avenues based on its established role in APAP-induced liver injury . As a potential biomarker, validation studies correlating serum/plasma BTF3L4 levels with liver injury severity across diverse patient populations would establish its clinical utility for early detection of drug-induced liver injury. Longitudinal studies tracking BTF3L4 expression during liver injury progression and resolution could determine its prognostic value for predicting outcomes. For therapeutic targeting, development of small molecule inhibitors that block BTF3L4 transcriptional activity or antisense oligonucleotides that reduce BTF3L4 expression would provide direct intervention strategies. Since BTF3L4 mediates liver injury through inflammatory responses and mitochondrial dysfunction, combination therapy approaches targeting BTF3L4 alongside anti-inflammatory or mitochondrial protective agents could yield synergistic benefits . High-throughput screening of compound libraries to identify molecules that modulate BTF3L4 expression or activity would expand the therapeutic arsenal. For precision medicine applications, investigating genetic variants affecting BTF3L4 expression or function could identify patient subpopulations at increased risk for drug-induced liver injury or those likely to respond to BTF3L4-targeted interventions. Development of liver-targeted delivery systems (nanoparticles, liposomes) for BTF3L4 modulators would enhance therapeutic specificity while minimizing off-target effects. Finally, exploring whether BTF3L4 plays similar pathogenic roles in other liver pathologies beyond APAP-induced injury (viral hepatitis, alcoholic/non-alcoholic steatohepatitis) would broaden its potential clinical applications as both biomarker and therapeutic target.

What experimental designs would best elucidate the regulatory mechanisms controlling BTF3L4 expression during liver injury?

To elucidate regulatory mechanisms controlling BTF3L4 expression during liver injury, comprehensive experimental designs spanning multiple investigative layers are necessary. Promoter analysis studies using reporter assays with serial deletions and mutations of the BTF3L4 promoter region would identify critical regulatory elements responsive to liver injury signals. Chromatin immunoprecipitation sequencing (ChIP-seq) to identify transcription factors binding to the BTF3L4 promoter during APAP-induced liver injury, followed by functional validation using siRNA knockdown of candidate factors, would establish key upstream regulators . Epigenetic profiling through bisulfite sequencing and chromatin accessibility assays (ATAC-seq) across different timepoints of liver injury would reveal whether DNA methylation or chromatin remodeling contributes to BTF3L4 expression changes. Investigation of post-transcriptional regulation through RNA immunoprecipitation to identify RNA-binding proteins interacting with BTF3L4 mRNA, and analysis of microRNAs potentially targeting BTF3L4 using prediction algorithms followed by luciferase reporter validations, would uncover additional regulatory layers. For signaling pathway involvement, systematic inhibition of major stress-responsive pathways (MAPK, NF-κB, ER stress) using pharmacological inhibitors or genetic approaches in APAP-treated hepatocytes, followed by BTF3L4 expression analysis, would identify pathway dependencies . Single-cell RNA sequencing of liver tissues during injury progression would reveal cell type-specific expression patterns and potential regulatory differences. Finally, transgenic mouse models with reporter genes driven by the BTF3L4 promoter would enable real-time visualization of expression dynamics during liver injury in vivo, while conditional knockout models would facilitate investigation of feedback mechanisms potentially involving BTF3L4 itself in regulating its own expression.

How do the expression patterns of BTF3L4 compare across different liver injury models?

This comparative analysis reveals that BTF3L4 upregulation is most pronounced and well-characterized in APAP-induced liver injury models, where its expression strongly correlates with established injury markers . While hypothesized patterns for other liver injury models are presented based on mechanistic similarities, dedicated studies investigating BTF3L4 expression across diverse pathological contexts remain necessary for comprehensive understanding of its role in liver pathophysiology.

What is the comparative efficacy of different antibody-based techniques for BTF3L4 detection?

This comparative analysis demonstrates that while each antibody-based technique offers distinct advantages, immunohistochemistry and immunofluorescence at dilutions of 1:50-1:100 have been most effectively utilized in BTF3L4 research for liver injury models . Western blotting provides essential validation of antibody specificity, while ELISA at high dilutions (1:40000) offers potential for biomarker development . The optimal technique selection depends on specific research questions, with consideration of sensitivity requirements and whether spatial information is critical to the experimental design.

What is the relationship between BTF3L4 expression and key pathological markers in APAP-induced liver injury?

This comprehensive analysis demonstrates that BTF3L4 expression strongly correlates with key indicators of hepatocellular damage, apoptosis, and inflammation in APAP-induced liver injury models . The temporal relationships suggest BTF3L4 functions as an early mediator of injury rather than merely a consequence, with particularly strong connections to mitochondrial dysfunction, ROS production, and subsequent apoptotic cell death . These correlations provide strong evidence for BTF3L4's central role in the pathogenesis of AILI and highlight its potential value as both a biomarker and therapeutic target.