ITGB4 Antibody

What is ITGB4 and why is it important in cellular research?

ITGB4 (integrin subunit beta 4) is a 202.2 kilodalton protein that forms part of the integrin α6β4 heterodimer, serving as a receptor for laminin. It plays a critical structural role in hemidesmosomes of epithelial cells and regulates keratinocyte polarity and motility . ITGB4's significance in research stems from its involvement in multiple cancer types, including breast cancer and lung adenocarcinoma, where it contributes to processes like drug resistance, cell migration, and invasion . For researchers, studying ITGB4 provides insights into fundamental cellular processes as well as potential therapeutic approaches for cancer treatment. Methodologically, this requires specific antibodies that can reliably detect ITGB4 across various experimental conditions and biological samples.

What are the common synonyms and identifiers for ITGB4 antibodies in scientific literature?

When searching scientific literature for ITGB4 antibodies, researchers should be aware of multiple nomenclatures. ITGB4 is commonly referenced as Integrin beta 4, CD104, GP150, or CD104 antigen . In antibody catalogs and publications, you might find it under any of these names. Additionally, when examining orthologs in animal models, researchers should search using species-specific terminology, as canine, porcine, monkey, mouse, and rat orthologs exist based on gene name . When citing ITGB4 in publications, it's recommended to include the primary term (ITGB4) along with a commonly recognized alternative (such as CD104) to ensure clarity across different research fields. This approach facilitates more comprehensive literature searches and improves reproducibility in experimental design.

How do I select the appropriate ITGB4 antibody for my specific research application?

Selecting the appropriate ITGB4 antibody requires consideration of multiple experimental parameters. First, determine your application needs (Western blotting, immunohistochemistry, flow cytometry, etc.), as different antibodies show variable performance across techniques . For instance, some antibodies like ab236251 are suitable for WB, IHC-P, and ICC/IF applications with human and mouse samples . Second, consider species reactivity—while many ITGB4 antibodies target human samples, cross-reactivity with mouse, rat, or other model organisms varies significantly . Third, evaluate the epitope location—antibodies targeting different regions of ITGB4 (such as the extracellular domain versus the cytoplasmic tail) may yield different results, particularly in functional studies. For example, some antibodies specifically recognize the 1400-1550 amino acid region . Finally, examine validation data, including positive controls in tissues known to express ITGB4 (epithelial cells, particularly in skin and lung) and negative controls. Published citations using specific antibody clones provide valuable insights into reliability and performance in contexts similar to your research question.

How does ITGB4 contribute to cancer drug resistance mechanisms?

ITGB4 promotes cancer drug resistance through multiple molecular pathways that enhance DNA damage repair and cell survival. In triple-negative breast cancer (TNBC), ITGB4 operates through the TNFAIP2/IQGAP1/RAC1 axis to confer resistance to DNA-damaging agents like epirubicin (EPI) and BMN . Mechanistically, ITGB4 interacts with TNFAIP2, which binds to IQGAP1 through its N-terminus (1-79 amino acids), subsequently activating RAC1. This activation enhances DNA damage repair, evidenced by decreased γH2AX and cleaved-PARP expression following treatment with chemotherapeutic agents . Additionally, ITGB4 promotes resistance to tamoxifen-induced apoptosis in breast cancer by activating the PI3K/AKT signaling pathway and confers resistance to anoikis through RAC1 activation . To investigate these mechanisms, researchers should employ ITGB4 knockdown strategies (siRNA, shRNA) followed by viability assays in the presence of chemotherapeutic agents, combined with assessment of DNA damage markers (γH2AX, 53BP1) and pathway-specific proteins (phosphorylated AKT, active RAC1) through Western blotting or immunofluorescence.

What is the expression pattern of ITGB4 across different cancer types and what are its prognostic implications?

ITGB4 exhibits differential expression patterns across cancer types, with significant upregulation observed in lung adenocarcinoma (LUAD) and triple-negative breast cancer (TNBC) compared to normal tissues . In LUAD, ITGB4 overexpression can be verified through analysis of The Cancer Genome Atlas (TCGA) data using tools like GEPIA2, with confirmation through immunohistochemistry using the Human Protein Atlas database . Prognostically, elevated ITGB4 levels predict adverse clinical outcomes in LUAD, as demonstrated by Kaplan-Meier survival analysis of patients stratified by ITGB4 expression (high vs. low, using median expression as the cutoff) . Methodologically, researchers should employ multiple approaches to assess ITGB4 expression, including qRT-PCR for mRNA levels, Western blotting for protein expression, and immunohistochemistry for tissue localization. For prognostic studies, it's crucial to use multivariate analysis that accounts for confounding factors such as tumor stage, grade, and patient demographics. Additionally, researchers should consider ITGB4's association with cancer stem cell markers, as ITGB4-positive cancer stem cells demonstrate specific properties that may influence disease progression and treatment response .

How can I effectively design ITGB4 knockdown experiments to study its role in cancer progression?

Designing effective ITGB4 knockdown experiments requires careful consideration of several methodological aspects. First, select appropriate cell models that naturally express high levels of ITGB4, such as A549 and PC9 lung adenocarcinoma cells or HCC1806 and HCC1937 triple-negative breast cancer cells . For transient knockdown, design at least 2-3 different siRNA sequences targeting distinct regions of ITGB4 mRNA to control for off-target effects. Alternatively, for stable knockdown, employ shRNA constructs delivered via lentiviral vectors . Confirm knockdown efficiency at both mRNA level (qRT-PCR) and protein level (Western blot) before proceeding with functional assays.

To comprehensively assess ITGB4's role, implement multiple functional assays: proliferation (MTT/CCK-8/BrdU incorporation), colony formation, apoptosis (Annexin V/PI staining, cleaved caspase-3/7 detection), migration (wound healing), and invasion (Transwell with Matrigel) . Include positive controls (genes known to affect these processes) and negative controls (non-targeting siRNA). For mechanistic insights, complement functional assays with signaling pathway analysis, focusing on RAC1 activation, PI3K/AKT signaling, and DNA damage repair markers (γH2AX) . Consider high-throughput approaches like RNA sequencing to identify global gene expression changes following ITGB4 knockdown, with subsequent pathway enrichment analysis to uncover broader biological processes affected .

What are the optimal conditions for Western blot detection of ITGB4?

Optimal Western blot detection of ITGB4 requires specific considerations due to its large size (202.2 kDa) and potential post-translational modifications. For protein extraction, use RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors if studying phosphorylated forms. Given ITGB4's high molecular weight, prepare 6-8% polyacrylamide gels or use gradient gels (4-15%) with extended separation times (100-120V for 2-3 hours) . For transfer, employ wet transfer systems at low voltage (30V) overnight at 4°C to efficiently transfer large proteins.

When selecting primary antibodies, those targeting the C-terminal region often provide better specificity, such as the rabbit polyclonal antibody ab236251 which recognizes amino acids 1400-1550 . Dilution ratios typically range from 1:500 to 1:1000, but should be optimized for each antibody . Extended primary antibody incubation (overnight at 4°C) generally yields better results. For visualization, secondary antibodies conjugated to HRP can be used at 1:5000 to 1:50000 dilutions , followed by enhanced chemiluminescence detection.

Appropriate positive controls include cell lines known to express high levels of ITGB4 (A549, MCF7) or tissues like skin and lung epithelia. For normalization, avoid typical housekeeping genes due to the size difference; instead, use total protein normalization methods like Ponceau S staining or loading controls in a similar molecular weight range. If studying cleaved fragments of ITGB4, which can occur during apoptosis or specific signaling events, note the expected molecular weights of these fragments in your experimental design and analysis.

How can I optimize immunohistochemistry protocols for ITGB4 detection in tissue samples?

Optimizing immunohistochemistry (IHC) for ITGB4 detection requires attention to several technical aspects. Begin with proper tissue fixation—10% neutral buffered formalin for 24-48 hours provides good antigen preservation while maintaining tissue architecture. Paraffin embedding should follow standard protocols, with sections cut at 4-5μm thickness . Antigen retrieval is critical for ITGB4 detection; heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 20 minutes provides good results, though optimal conditions should be determined empirically for each antibody.

When selecting primary antibodies, those validated specifically for IHC-P applications should be prioritized, such as the rabbit polyclonal antibody ab236251 . Typical working dilutions range from 1:100 to 1:500, with overnight incubation at 4°C. For detection systems, polymer-based methods often provide better sensitivity than avidin-biotin complexes. Counterstaining with hematoxylin should be brief to avoid masking specific ITGB4 staining.

For validation, include positive control tissues known to express ITGB4 (skin, particularly in the basal layer of the epidermis; bronchial epithelium). Negative controls should include both technical controls (primary antibody omission) and biological controls (tissues known not to express ITGB4). ITGB4 typically shows membrane staining, particularly at the basal surface of epithelial cells where hemidesmosomes form. In cancer tissues, altered localization patterns (cytoplasmic, diffuse membrane) may have biological significance and should be noted. For quantification, consider using digital pathology approaches with appropriate software to score staining intensity and percentage of positive cells, generating H-scores or Allred scores for statistical comparisons.

What are the technical considerations for immunofluorescence detection of ITGB4 in cell culture models?

Immunofluorescence (IF) detection of ITGB4 in cultured cells requires specific technical considerations to achieve optimal visualization of its characteristic distribution patterns. Begin with appropriate cell models—epithelial cell lines (MCF10A, HaCaT, bronchial epithelial cells) typically express ITGB4 at hemidesmosomes along the basal surface when grown on appropriate substrates . Consider growing cells on laminin-coated surfaces to enhance ITGB4 clustering and hemidesmosome formation, making detection more distinct.

For fixation, 4% paraformaldehyde (15 minutes at room temperature) preserves most epitopes while maintaining cellular architecture. Avoid methanol fixation which can disrupt membrane proteins unless specifically recommended for your antibody. Permeabilization should be gentle (0.1-0.2% Triton X-100 for 5-10 minutes) to access intracellular epitopes without disrupting membrane structures.

Primary antibody selection should prioritize those validated for ICC/IF applications, such as the rabbit polyclonal antibody ab236251 . Working dilutions typically range from 1:100 to 1:500, with overnight incubation at 4°C. For costaining experiments, combine ITGB4 detection with markers of hemidesmosomes (plectin, BP180, BP230) or basement membrane components (laminin-332) to visualize functional complexes. Select secondary antibodies with fluorophores appropriate for your microscopy setup, avoiding spectral overlap if performing multilabel experiments.

Counterstain nuclei with DAPI or Hoechst, and consider phalloidin staining to visualize F-actin, providing context for ITGB4 localization relative to the cytoskeleton. For advanced applications, super-resolution microscopy techniques (STORM, PALM, SIM) can resolve the detailed organization of ITGB4 within hemidesmosomes, which are typically below the diffraction limit of conventional microscopy.

How does ITGB4 interact with growth factor receptors to modulate cancer cell signaling?

ITGB4 forms complex interactions with multiple growth factor receptors, creating signaling hubs that modulate cancer cell behavior. The integrin α6β4 heterodimer directly binds to neuregulin-1 (NRG1) through its EGF domain, and this interaction is essential for proper NRG1-ERBB signaling . Similarly, α6β4 binds directly to insulin-like growth factors IGF1 and IGF2, which is crucial for activating their downstream signaling cascades . In non-small cell lung cancer, the α6β4 heterodimer interacts with the receptor tyrosine kinase MET to promote tumor invasion .

Methodologically, these interactions can be studied using multiple approaches. Co-immunoprecipitation experiments using antibodies against ITGB4 can identify associated growth factor receptors in cell lysates, with reverse co-IP confirming bidirectional interactions. Proximity ligation assays provide visualization of protein-protein interactions in situ with subcellular resolution. For functional studies, researchers should compare signaling pathway activation (phosphorylation of downstream effectors) in response to growth factors (NRG1, IGF1, IGF2) in wild-type versus ITGB4-knockdown cells.

The biological significance of these interactions lies in their ability to amplify growth factor signaling. When ITGB4 and growth factor receptors co-localize, they can enhance pathway activation through mechanisms including receptor clustering, prevention of internalization, or direct modulation of kinase activity. This cross-talk contributes to cancer cell proliferation, survival, and therapeutic resistance, making these interactions potential targets for combination therapy approaches.

What is the role of ITGB4 in regulating RAC1 activation and how does this impact cancer cell behavior?

ITGB4 regulates RAC1 activation through a complex molecular pathway involving multiple interacting proteins. In triple-negative breast cancer, ITGB4 activates RAC1 via the TNFAIP2/IQGAP1 axis . Mechanistically, ITGB4 interacts with TNFAIP2, which then binds to IQGAP1 through its N-terminal domain (amino acids 1-79). IQGAP1, despite lacking the typical GTPase-activating function due to the absence of an arginine in its GTPase binding domain, increases RAC1 activity rather than promoting GTP hydrolysis . This activation is critical because active RAC1 promotes DNA damage repair, conferring resistance to chemotherapeutic agents that induce DNA damage .

To study this pathway, researchers can employ multiple methodological approaches. RAC1 activation can be measured using pull-down assays with the p21-binding domain of PAK1, which specifically binds active (GTP-bound) RAC1. Alternatively, FRET-based biosensors allow real-time visualization of RAC1 activation in living cells. The interaction between ITGB4, TNFAIP2, and IQGAP1 can be confirmed through co-immunoprecipitation assays and mapping of binding domains using truncated protein constructs .

The biological impact of ITGB4-mediated RAC1 activation extends beyond drug resistance. RAC1 is a key regulator of actin cytoskeleton dynamics, influencing cell migration, invasion, and metastasis. Additionally, RAC1 contributes to cancer cell survival by activating anti-apoptotic signaling pathways. Targeting this ITGB4/TNFAIP2/IQGAP1/RAC1 axis represents a potential strategy to overcome chemoresistance in TNBC and possibly other cancer types where ITGB4 is overexpressed .

How does ITGB4 regulate gene expression patterns in cancer cells?

ITGB4 exerts profound effects on gene expression patterns in cancer cells, modulating multiple cellular processes that contribute to malignant phenotypes. High-throughput sequencing of ITGB4-knockdown lung adenocarcinoma cells reveals that ITGB4 upregulates genes enriched in metabolism and related pathways, while downregulating genes involved in cell cycle regulation . This dual regulatory role suggests that ITGB4 promotes metabolic adaptations while simultaneously affecting proliferative capacity.

Methodologically, researchers investigating ITGB4's transcriptional effects should employ RNA sequencing or microarray analysis comparing control versus ITGB4-knockdown or ITGB4-overexpressing cells. Differential expression analysis should be followed by pathway enrichment analysis using tools like KEGG, GO, or Reactome to identify biological processes affected. Validation of key differentially expressed genes should be performed using qRT-PCR, and protein-level confirmation by Western blotting is recommended for selected targets.

The mechanisms through which ITGB4 modulates gene expression likely involve activation of transcription factors downstream of signaling pathways like PI3K/AKT , which can regulate metabolic gene expression. Additionally, ITGB4-mediated activation of RAC1 may influence gene expression through effects on cytoskeletal dynamics and nuclear translocation of transcriptional regulators. For more direct evidence of transcriptional regulation, chromatin immunoprecipitation followed by sequencing (ChIP-seq) of transcription factors suspected to be downstream of ITGB4 signaling can identify specific genomic regions and target genes affected. Understanding these transcriptional networks is crucial for developing strategies to counteract ITGB4-driven oncogenic programs in cancer.

How can ITGB4 be targeted therapeutically in cancer treatment strategies?

Targeting ITGB4 therapeutically represents a promising approach for cancer treatment, particularly for tumors with ITGB4 overexpression like triple-negative breast cancer and lung adenocarcinoma . Several strategic approaches can be implemented, each with specific methodological considerations. First, neutralizing antibodies against the extracellular domain of ITGB4 can block its interaction with laminin and other binding partners, potentially reducing downstream signaling. Such antibodies should be evaluated for their ability to induce internalization of the receptor and for potential antibody-dependent cellular cytotoxicity.

Second, small molecule inhibitors targeting the intracellular signaling domains of ITGB4 could disrupt its interactions with TNFAIP2 and IQGAP1, thereby preventing RAC1 activation and associated drug resistance mechanisms . High-throughput screening approaches using protein-protein interaction assays can identify candidate molecules that disrupt these specific interactions. Third, antisense oligonucleotides or siRNA-based therapeutics delivered via nanoparticles could downregulate ITGB4 expression, mimicking the effects observed in knockdown experiments that attenuate proliferation, migration, and invasion .

Combination therapies targeting ITGB4 alongside conventional chemotherapeutics show particular promise, as ITGB4 inhibition could sensitize resistant cancer cells to DNA-damaging agents by impairing repair mechanisms . For preclinical development, appropriate models include patient-derived xenografts that maintain ITGB4 expression patterns and associated stromal interactions. Efficacy should be assessed not only by tumor growth inhibition but also by examining DNA damage markers, apoptotic indices, and metastatic burden. Potential challenges include compensatory upregulation of other integrin subunits and toxicity to normal epithelial tissues where ITGB4 plays important structural roles, necessitating careful therapeutic window determination.

What approaches can be used to study the role of ITGB4 in cancer stem cell populations?

Studying ITGB4 in cancer stem cell (CSC) populations requires specialized methodological approaches due to the heterogeneity and relative rarity of these cells. Research has shown that ITGB4-positive cancer stem cells reside in an intermediate epithelial/mesenchymal phenotypic state, and ITGB4 expression enables stratification of mesenchymal-like triple-negative breast cancer cells . Additionally, ITGB4 expression is significantly higher in ALDH-high breast cancer and head and neck cancer cells compared to ALDH-low populations .

To isolate ITGB4-positive CSCs, fluorescence-activated cell sorting (FACS) using anti-ITGB4 antibodies conjugated to fluorophores can be combined with established CSC markers like ALDH activity (measured by the ALDEFLUOR assay) or CD44+/CD24- phenotype. Single-cell RNA sequencing of sorted populations provides insights into heterogeneity within ITGB4+ cells and identifies co-expression patterns with stemness-associated genes.

Functional characterization should include sphere-forming assays under low-attachment conditions, limiting dilution assays to quantify CSC frequency, and in vivo tumor initiation studies using serial transplantation in immunodeficient mice to assess self-renewal capacity. The contribution of ITGB4 to stemness can be evaluated through knockdown or overexpression experiments followed by these functional assays. Additionally, drug resistance profiles of ITGB4+ versus ITGB4- cells should be compared using dose-response curves for clinically relevant chemotherapeutics.

To understand mechanisms underlying ITGB4's role in CSCs, investigate its interaction with known stemness-promoting pathways like Wnt/β-catenin, Notch, and Hedgehog. Chromatin immunoprecipitation sequencing (ChIP-seq) for stemness-associated transcription factors in ITGB4+ cells can identify direct transcriptional targets. Finally, spatial organization of ITGB4+ CSCs within tumors should be examined using multiplexed immunofluorescence to understand their relationship with the tumor microenvironment, particularly with cancer-associated fibroblasts that have been shown to receive ITGB4 via exosomes .

How can advanced microscopy techniques enhance our understanding of ITGB4 dynamics in cancer progression?

Advanced microscopy techniques offer unprecedented insights into ITGB4 dynamics during cancer progression, revealing spatial, temporal, and functional aspects not accessible with conventional methods. Super-resolution microscopy approaches such as Structured Illumination Microscopy (SIM), Stimulated Emission Depletion (STED), and Single-Molecule Localization Microscopy (STORM/PALM) can resolve ITGB4 organization within hemidesmosomes at nanoscale resolution (20-100 nm), revealing how these structures are dismantled during epithelial-to-mesenchymal transition in cancer cells .

Live-cell imaging combined with fluorescent protein tagging (ITGB4-GFP) enables tracking of receptor dynamics, endocytosis, and recycling in real-time. This approach can reveal how ITGB4 trafficking changes in response to extracellular matrix composition, growth factor stimulation, or therapeutic interventions. Fluorescence Recovery After Photobleaching (FRAP) and photoactivation techniques provide quantitative measurements of ITGB4 mobility and turnover rates at the plasma membrane, parameters that often change during malignant transformation.

For protein-protein interactions, Förster Resonance Energy Transfer (FRET) microscopy between ITGB4 and its binding partners (TNFAIP2, IQGAP1, growth factor receptors) provides spatial maps of interaction sites within cells . Proximity Ligation Assay (PLA) offers an alternative approach with high sensitivity for detecting endogenous protein interactions in fixed specimens. To study ITGB4 in the tumor microenvironment, intravital microscopy in window chamber models allows visualization of ITGB4-expressing cells during invasion and metastasis in living animals.

Correlative Light and Electron Microscopy (CLEM) combines the molecular specificity of fluorescence imaging with the ultrastructural context of electron microscopy, revealing how ITGB4-containing hemidesmosomes are remodeled at the nanoscale during cancer progression. Finally, multiplexed imaging approaches using cyclic immunofluorescence or mass cytometry imaging allow simultaneous visualization of ITGB4 with dozens of other markers, providing comprehensive phenotypic profiles of ITGB4-expressing cells within heterogeneous tumors. These advanced techniques collectively provide a multi-scale understanding of ITGB4 biology that can inform more effective therapeutic strategies.