TGFB1I1 Antibody

What is TGFB1I1 and what cellular functions should researchers consider when selecting antibodies?

TGFB1I1, also known as HIC-5 (Hydrogen Peroxide-Inducible Clone-5), functions as a molecular adapter that coordinates multiple protein-protein interactions at focal adhesion complexes and in the nucleus. This protein links various intracellular signaling modules to plasma membrane receptors and regulates the Wnt and TGFB signaling pathways . It may also regulate SLC6A3 and SLC6A4 targeting to the plasma membrane, thus modulating their activity .

In the nucleus, TGFB1I1 functions as a nuclear receptor coactivator regulating glucocorticoid, androgen, mineralocorticoid, and progesterone receptor transcriptional activity . Research has established its role in:

• Cell growth, proliferation, migration, and differentiation

• Cellular senescence

• Focal adhesion formation

• Epithelial-mesenchymal transition (EMT)

• TGF-β signaling pathway regulation

• Transcriptional co-activation

When selecting antibodies, researchers should consider which domain or region of TGFB1I1 is relevant to their specific research question, as different antibodies target different epitopes.

Which applications are most reliable for TGFB1I1 antibodies in research?

TGFB1I1 antibodies have been validated for multiple applications, with varying degrees of reliability:

Detection in Western blot and immunofluorescence have shown the most consistent results across multiple studies. For example, Anti-Human HIC5/TGFB1I1 antibody successfully detected TGFB1I1 in MCF-7 human breast cancer cell line and HepG2 human hepatocellular carcinoma cell line at approximately 50 kDa .

What are the critical validation steps researchers should perform before using TGFB1I1 antibodies?

Proper validation is essential for ensuring experimental reproducibility when working with TGFB1I1 antibodies:

• Positive and negative controls: Use cell lines known to express (e.g., MCF-7, HepG2, PC-3) or not express TGFB1I1.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to confirm specificity.

• Knockout/knockdown validation: Compare antibody reactivity in TGFB1I1 wildtype versus knockout/knockdown samples.

• Cross-reactivity assessment: For antibodies claiming multi-species reactivity, verify performance across species (predicted reactivity: Cow: 100%, Dog: 93%, Goat: 86%, Guinea Pig: 100%, Horse: 100%, Human: 100%, Mouse: 100%, Rabbit: 100%, Rat: 100%) .

• Application-specific validation: Verify antibody performance specifically for your intended application (WB, IHC, IF, etc.).

Research by Liang et al. demonstrated that TGFB1I1 knockdown resulted in decreased EMT marker expression in urothelial carcinoma cells, providing an excellent system for antibody validation .

How should researchers interpret subcellular localization patterns when using TGFB1I1 antibodies?

TGFB1I1 exhibits dynamic subcellular localization that changes based on cellular conditions:

• Focal adhesions: Primary localization under normal conditions .

• Cytoplasmic: Often observed in unstimulated cells .

• Nuclear: Increases upon stimulation (e.g., with TGF-β1 or BMP-4) .

For example, in PC-3 human prostate cancer cells, TGFB1I1 was detected primarily in the cytoplasm when unstimulated, but showed increased nuclear localization after stimulation with 10 ng/mL Recombinant Human BMP-4 . This shuttling between compartments is functionally significant as it relates to TGFB1I1's dual roles in adhesion complexes and as a transcriptional co-regulator.

When interpreting localization patterns, researchers should:

• Use counterstains (e.g., DAPI for nucleus, phalloidin for actin cytoskeleton)

• Compare unstimulated versus stimulated conditions

• Consider fixation methods, as they can affect apparent localization

• Document exposure time and imaging parameters for reproducibility

What factors affect TGFB1I1 antibody performance in Western blot applications?

Multiple factors can influence TGFB1I1 detection by Western blot:

Researchers have successfully detected TGFB1I1 in MCF-7 and HepG2 cell lines using PVDF membrane probed with 1 μg/mL of Goat Anti-Human HIC5/TGFB1I1 Antigen Affinity-purified Polyclonal Antibody followed by HRP-conjugated Anti-Goat IgG Secondary Antibody .

How can researchers distinguish between TGFB1I1 isoforms and post-translational modifications?

TGFB1I1 exists in multiple isoforms and undergoes various post-translational modifications that can affect antibody recognition:

• Isoform discrimination:

  • Use isoform-specific antibodies targeting unique regions

  • Compare antibodies recognizing different domains (N-terminal vs. Middle region vs. C-terminal)

  • Perform immunoprecipitation followed by mass spectrometry

• Post-translational modifications (PTMs):

  • Phosphorylation may alter apparent molecular weight

  • Use phospho-specific antibodies when studying signaling events

  • Treat lysates with phosphatase to confirm phosphorylation status

• Size verification:

  • TGFB1I1 typically appears at approximately 50 kDa

  • Higher molecular weight bands may indicate PTMs or protein complexes

  • Lower molecular weight bands could represent degradation products or alternative isoforms

For transcript variants, researchers can reference specific variant information (e.g., Transcript Variant 2, Transcript Variant 3) as noted in product descriptions .

What controls are essential when using TGFB1I1 antibodies in immunohistochemistry?

When performing immunohistochemistry with TGFB1I1 antibodies, several controls are essential:

• Positive tissue control: Use tissues known to express TGFB1I1, such as:

  • Prostate tissue (PC-3 cells have shown positive staining)

  • Breast cancer tissue (MCF-7 cells express TGFB1I1)

  • Urothelial carcinoma samples (shown to overexpress TGFB1I1)

• Negative tissue control: Include tissues known not to express TGFB1I1

• Antibody controls:

  • Primary antibody omission control

  • Isotype control (using matched IgG at the same concentration)

  • Peptide competition control (pre-incubating antibody with immunizing peptide)

• Signal verification controls:

  • Use of alternative antibodies targeting different epitopes

  • Correlation with mRNA expression (e.g., by RT-PCR or ISH)

  • Comparison with TGFB1I1 knockdown or knockout tissues

In urothelial carcinoma studies, overexpression of TGFB1I1 showed significant correlation with advanced tumor stage, papillary configuration, and frequent mitosis, making these parameters useful for validating staining patterns .

How should researchers approach troubleshooting non-specific binding with TGFB1I1 antibodies?

Non-specific binding is a common challenge when working with antibodies. For TGFB1I1 antibodies, consider these troubleshooting approaches:

• Optimization of antibody concentration:

  • Perform a dilution series (e.g., 0.1, 0.5, 1, 5, 10 μg/mL)

  • Balance signal intensity with background

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Increase blocking time (1-2 hours at room temperature)

  • Add 0.1-0.3% Triton X-100 for membrane permeabilization in IF

• Washing optimization:

  • Increase wash duration and number of washes

  • Add 0.05-0.1% Tween-20 to wash buffers

• Sample preparation:

  • Test different fixation methods (PFA, methanol, acetone)

  • Optimize antigen retrieval (citrate buffer, EDTA buffer, enzymatic retrieval)

• Secondary antibody considerations:

  • Use highly cross-adsorbed secondary antibodies

  • Consider fluorophore brightness and spectral overlap in IF

  • Minimize secondary antibody concentration

For validating specificity, researchers have used BMP-4 stimulation to induce nuclear translocation of TGFB1I1 in PC-3 cells, which provides a functional verification of antibody specificity .

What methodological approaches enable studying TGFB1I1's role in epithelial-mesenchymal transition (EMT)?

TGFB1I1 has been implicated in epithelial-mesenchymal transition (EMT), particularly in cancer progression. To investigate this role:

• Knockdown/overexpression studies:

  • Use siRNA/shRNA to knockdown or expression vectors to overexpress TGFB1I1

  • Monitor EMT markers before and after manipulation

  • Research by Liang et al. showed that TGFB1I1 knockdown decreased EMT markers in urothelial carcinoma cells

• Co-localization studies:

  • Perform dual immunofluorescence for TGFB1I1 and EMT markers (vimentin, E-cadherin, N-cadherin)

  • Use confocal microscopy for high-resolution analysis

  • Quantify co-localization using appropriate software

• TGF-β stimulation time course:

  • Treat cells with TGF-β1 and collect samples at different time points

  • Monitor TGFB1I1 expression, localization, and phosphorylation status

  • Correlate with EMT progression markers

• Focal adhesion dynamics:

  • Use live-cell imaging with fluorescently-tagged TGFB1I1

  • Monitor focal adhesion turnover during EMT

  • Correlate with cell migration capacity

• Protein-protein interaction analysis:

  • Perform co-immunoprecipitation of TGFB1I1 with focal adhesion proteins

  • Use proximity ligation assay to visualize interactions in situ

  • Apply FRET/BRET techniques for real-time interaction studies

Research has demonstrated that TGFB1I1 promotes focal adhesion formation, contributing to EMT with actin cytoskeleton and vimentin reorganization through TGFB1I1 regulation .

How can researchers effectively use TGFB1I1 antibodies to investigate its interactions with nuclear receptors?

TGFB1I1 functions as a nuclear receptor coactivator. To study these interactions:

• Nuclear extraction protocols:

  • Use validated nuclear/cytoplasmic fractionation kits

  • Verify fraction purity with compartment-specific markers

  • Optimize extraction conditions to preserve protein-protein interactions

• Co-immunoprecipitation (Co-IP) approaches:

  • Immunoprecipitate TGFB1I1 and probe for associated nuclear receptors

  • Perform reverse Co-IP (pull down receptors, probe for TGFB1I1)

  • Use crosslinking to stabilize transient interactions

• Chromatin immunoprecipitation (ChIP):

  • Perform ChIP with TGFB1I1 antibodies to identify binding sites

  • Perform sequential ChIP (first for receptor, then for TGFB1I1) to confirm co-occupancy

  • Correlate with transcriptional activity of target genes

• Proximity-based assays:

  • BioID or APEX2 proximity labeling with TGFB1I1 as bait

  • Proximity ligation assay (PLA) for in situ visualization

  • FRET/BRET for real-time interaction dynamics

• Functional transcription assays:

  • Luciferase reporter assays with nuclear receptor response elements

  • Compare wildtype TGFB1I1 versus mutants lacking interaction domains

  • Correlate with endogenous target gene expression by RT-qPCR

Studies have shown that TGFB1I1 interacts with androgen receptor (AR) as a coactivator (ARA55), suggesting its importance in AR-regulated transcriptional activity .

What strategies can researchers employ to study TGFB1I1's role in the TGF-β signaling pathway using antibody-based approaches?

TGFB1I1 is intricately linked to TGF-β signaling. To investigate this relationship:

• Phosphorylation dynamics:

  • Monitor TGFB1I1 phosphorylation status after TGF-β1 stimulation

  • Use phospho-specific antibodies if available

  • Correlate with SMAD2/3 phosphorylation kinetics

• Signaling pathway analysis:

  • Evaluate effects of TGFB1I1 knockdown on TGF-β signaling components

  • Research has shown that TGF-β1 suppresses proinflammatory gene expression while potently inducing contractile genes in vascular smooth muscle cells through a SMAD4-dependent canonical pathway

  • This process involves TGFB1I1 as detected by antibody-based methods

• Complex formation dynamics:

  • Study interaction of TGFB1I1 with SMAD proteins using Co-IP

  • Perform size exclusion chromatography followed by Western blot

  • Use BioID or APEX2 proximity labeling to identify novel interactors

• Subcellular trafficking:

  • Track TGFB1I1 movement after TGF-β stimulation using IF

  • Perform live-cell imaging with fluorescently-tagged TGFB1I1

  • Correlate localization changes with signaling events

• Target gene regulation:

  • Perform ChIP-seq with TGFB1I1 antibodies

  • Compare binding patterns with and without TGF-β stimulation

  • Correlate with transcriptional changes by RNA-seq

Research has demonstrated that TGF-β1 suppression of VSMC proinflammatory gene expression is mediated partially through blockade of both STAT3 and NF-κB pathways, with TGFB1I1 detection serving as a key marker in these studies .

How can researchers investigate TGFB1I1's role in retinal development and visual adaptation using antibody-based techniques?

Recent research has implicated TGFB1I1 in retinal circuitry development. To study this:

• Comparative analysis in wildtype versus knockout models:

  • Studies in Tgfb1i1−/− mice showed more Pax6 α-GFP-positive cells than wildtype littermates

  • This approach requires specific antibodies against TGFB1I1 and retinal cell markers

• Cell fate determination studies:

  • Use TGFB1I1 antibodies alongside markers for amacrine and bipolar cells

  • Research showed Tgfb1i1−/− mouse retinas have fewer Vsx1-positive OFF bipolar cells without significant changes in G0α-positive ON bipolar cells

• Molecular complex analysis:

  • Study the LIM protein complex (Tgfb1i1-Lhx3-Isl1) using co-immunoprecipitation

  • Investigate the interaction with Pax6 α-enhancer activity

  • Research suggests Tgfb1i1 forms a hetero-tetrameric complex that represses the Pax6 α-enhancer

• Regulatory network mapping:

  • Use ChIP-seq with TGFB1I1 antibodies to identify binding sites in retinal cells

  • Compare with transcriptomic changes in knockout models

  • Correlate with functional visual adaptations

• Visual response testing:

  • Mice lacking Tgfb1i1 showed stronger responses to light, comparable to photosensitivity in humans

  • Correlate protein expression patterns (detected by antibodies) with electrophysiological measurements

This research demonstrates how antibody-based detection of TGFB1I1 and associated proteins can reveal fundamental mechanisms in neural development and sensory processing.

What considerations are important when developing TCR-like antibodies targeting TGFB1I1-derived peptides presented by MHC?

For researchers interested in developing T-cell receptor (TCR)-like antibodies targeting TGFB1I1-derived peptides:

• Peptide selection criteria:

  • Identify TGFB1I1 peptides with high predicted binding affinity to MHC molecules

  • Focus on peptides from functionally important domains

  • Consider peptides differentially presented in disease states

• Antibody development approaches:

  • Phage display technology has successfully generated TCR-like antibodies

  • Different formats (Fab fragments, scFv, full IgG) have distinct advantages

  • TCR-like antibodies combine the recognition of intracellular proteins with the therapeutic potency of monoclonal antibodies

• Specificity verification:

  • Test binding to target peptide-MHC complex versus similar complexes

  • Research indicates TCR-like antibodies may have off-target peptides that share residues at major contacts

  • Some TCR-like antibodies bind only to specific regions of the peptide (e.g., N-terminal or C-terminal residues)

• Functional applications:

  • Consider development into CAR-T cell therapies (as demonstrated for other targets)

  • Evaluate potential for targeted drug delivery

  • Assess applicability for diagnostic imaging

• Technical challenges:

  • Production techniques for TCR-like antibodies are often proprietary

  • May require specialized expertise in both immunology and structural biology

  • Validation requires complex assays to confirm peptide-MHC specificity

Research has shown that TCR-like antibodies can be engineered into chimeric antigen receptors (CARs) for targeting intracellular antigens presented by MHC molecules, potentially opening new therapeutic avenues .

How can multidimensional imaging approaches enhance our understanding of TGFB1I1 function using antibody-based detection?

Advanced imaging methods can provide unprecedented insights into TGFB1I1 biology:

• Super-resolution microscopy:

  • Use STORM, PALM, or STED to visualize TGFB1I1 within focal adhesion structures

  • Achieve 10-20 nm resolution to map precise localization

  • Combine with other focal adhesion proteins for nanoscale architecture analysis

• Live-cell dynamics:

  • Apply FRAP (Fluorescence Recovery After Photobleaching) to study TGFB1I1 mobility

  • Use fluorescently-tagged TGFB1I1 with antibody validation

  • Record real-time translocation during signaling events

• Intravital imaging:

  • Utilize transparent tissue preparations (CLARITY, CUBIC)

  • Apply antibodies for whole-tissue TGFB1I1 distribution analysis

  • Correlate with physiological or pathological processes in vivo

• Correlative light-electron microscopy (CLEM):

  • Combine immunofluorescence with electron microscopy

  • Locate TGFB1I1 at ultrastructural level

  • Reveal association with specific cellular structures

• Multiplexed imaging:

  • Employ cyclic immunofluorescence or mass cytometry (CyTOF) imaging

  • Simultaneously detect TGFB1I1 with dozens of other markers

  • Create comprehensive maps of protein networks in different cellular states

These approaches can be particularly valuable for understanding the dynamic role of TGFB1I1 in processes like focal adhesion formation during cell migration and epithelial-mesenchymal transition, as observed in urothelial carcinoma research .

What methodological challenges exist when studying TGFB1I1 in different model organisms using antibody-based approaches?

Working with TGFB1I1 across species presents several challenges:

• Cross-species reactivity verification:

  • While many antibodies claim multi-species reactivity, performance varies significantly

  • Predicted reactivity (e.g., Cow: 100%, Dog: 93%, Human: 100%, Mouse: 100%, Rat: 100%) requires experimental verification

  • Sequence comparison between target regions in different species is essential

• Model-specific considerations:

  • Mouse: Most TGFB1I1 studies use mouse models, with good antibody availability

  • Rat: Several validated antibodies available

  • Non-mammalian models: Limited validation (e.g., Pogona vitticeps/bearded dragon TGFB1I1)

• Developmental timing:

  • TGFB1I1 expression varies during development

  • Studies in mouse retina showed developmental stage-specific effects

  • Antibody detection sensitivity must match expression levels

• Tissue processing differences:

  • Fixation protocols may need species-specific optimization

  • Antigen retrieval requirements differ between tissues and species

  • Background autofluorescence varies significantly across species

• Functional validation strategies:

  • Knockout models available in mouse (Tgfb1i1−/−) but limited in other species

  • CRISPR-based approaches allow targeted editing across species

  • Antibody-based detection crucial for validating genetic manipulations