AJUBA Antibody, HRP conjugated

What is AJUBA protein and why is it significant in cellular research?

AJUBA functions as an adapter or scaffold protein that participates in the assembly of numerous protein complexes and is involved in several cellular processes. It contributes to the linking and strengthening of epithelial cell-cell junctions by connecting adhesive receptors to the actin cytoskeleton . AJUBA plays an important role in regulating AURKA kinase activity for mitotic commitment and modulates IL-1-induced NFKB1 activation by influencing multiprotein signaling complex assembly . Recent studies have identified AJUBA as a novel negative regulator of p53, inhibiting apoptosis induced by chemotherapy drugs in a negative feedback manner . Its involvement in cancer progression, particularly colorectal cancer, makes it a significant target for ongoing research.

Which cellular compartments typically contain AJUBA protein?

AJUBA is primarily found in both cytoplasmic and nuclear compartments, as it shuttles between these locations to perform various functions . In epithelial cells, AJUBA localizes to cell-cell junctions where it contributes to junction stability . When investigating AJUBA's different functions, consider performing cellular fractionation to analyze its distribution between compartments. For immunofluorescence applications, validated protocols have successfully detected AJUBA in PFA-fixed, Triton X-100 permeabilized cells .

How does AJUBA contribute to cancer chemoresistance mechanisms?

AJUBA has been identified as a negative regulator of p53 that inhibits apoptosis induced by chemotherapy drugs through a negative feedback mechanism . Research shows that AJUBA:

• Forms a complex with p53 and MDM2

• Enhances p53/MDM2 interaction

• Promotes p53 degradation through the ubiquitin-proteasome pathway

In colorectal cancer cells, overexpression of AJUBA decreases p53 levels and its downstream target genes (p21 and BAX), while knockdown of AJUBA significantly increases p53 levels . Notably, AJUBA does not influence p53 transcription but rather regulates its post-transcriptional stability. Chemotherapeutic drugs (including 5-fluorouracil, oxaliplatin, adriamycin, and etoposide) significantly induce AJUBA expression in a p53-dependent manner, creating a negative feedback loop . This mechanism may explain why high AJUBA expression correlates with poorer survival in colorectal cancer patients.

What is known about AJUBA's role in regulating epithelial cell junctions?

AJUBA plays a critical role in maintaining epithelial cell-cell junction integrity through multiple mechanisms:

• It interacts with the Cdc42 GTPase activating protein CdGAP at cell-cell contacts

• While AJUBA does not recruit CdGAP to junctions, it controls CdGAP residence at cell-cell adhesion sites

• AJUBA binding inhibits CdGAP activity, thus maintaining junctional stability

• AJUBA interacts with Rac1 and CdGAP via distinct domains, potentially bringing them in close proximity at junctions to facilitate activity regulation

Research has shown that gain-of-function CdGAP mutants found in Adams-Oliver Syndrome patients strongly destabilize cell-cell contacts, and CdGAP mRNA levels are inversely correlated with E-cadherin protein expression in different cancers . This indicates AJUBA's important role in preserving epithelial tissue architecture both in normal homeostasis and in pathological conditions.

How does AJUBA participate in transcriptional regulation?

AJUBA functions as an HDAC-dependent corepressor for a subset of target genes through several mechanisms:

• It forms complexes with histone deacetylases HDAC1, HDAC2, and HDAC3

• Active histone deacetylase activity co-immunoprecipitates with AJUBA

• AJUBA acts as a transcriptional corepressor for SNAI1 and SNAI2/SLUG-dependent repression of E-cadherin transcription

• It functions as a corepressor for specific Growth factor independent-1 (Gfi1) target genes

• AJUBA positively regulates microRNA (miRNA)-mediated gene silencing

Experimental evidence from co-immunoprecipitation, gel filtration, and histone deacetylase activity assays all support the existence of an endogenous AJUBA·Gfi1·HDAC multiprotein complex . Mechanistically, AJUBA's LIM domains directly bind to Gfi1, though this association is not SNAG domain-dependent .

What are the optimal Western blot conditions for AJUBA detection using HRP-conjugated antibodies?

For optimal AJUBA detection using HRP-conjugated antibodies in Western blot, implement the following protocol:

When interpreting results, expect the primary AJUBA band at 55-60 kDa, though additional bands may appear. The identity of a band at approximately 30 kDa observed in some experiments remains unknown .

How can I design co-immunoprecipitation experiments to study AJUBA-mediated protein complexes?

To effectively investigate AJUBA-mediated protein complexes through co-immunoprecipitation:

• Sample preparation:

  • For cytoplasmic complexes: Use NP-40 buffer (0.5% NP-40, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA)

  • For nuclear complexes: Perform nuclear extraction followed by lysis in a buffer containing 20 mM HEPES pH 7.9, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 25% glycerol

  • Always include protease inhibitors and phosphatase inhibitors

• Critical controls:

  • Input sample (5-10% of lysate used for IP)

  • Isotype-matched IgG from the same species as the AJUBA antibody

  • Related protein control (e.g., antibodies against LPP)

  • Reciprocal co-IP with antibodies against each protein in the suspected complex

• For studying specific AJUBA complexes:

  • p53-MDM2 complex: Use HCT116 or RKO colorectal cancer cells

  • HDAC complexes: Use Jurkat cells for nuclear extraction

  • CdGAP interaction: Use epithelial cell models

• Advanced validation approaches:

  • Sequential co-IP for multi-protein complexes (AJUBA-p53-MDM2)

  • In vitro GST-pull down assays to confirm direct interactions

  • Domain mapping using truncation mutants of AJUBA (Pre-LIM vs. LIM domains)

Research has demonstrated that AJUBA interacts with p53 through its C-terminal LIM domain, binding specifically to the DNA-binding domain (DBD) of p53 . Similar domain-specific interactions have been mapped for other AJUBA binding partners.

What approaches can I use to study AJUBA's role in histone deacetylation processes?

To investigate AJUBA's function in histone deacetylation, implement these methodological approaches:

• Histone deacetylase activity assay:

  • Immunoprecipitate AJUBA from nuclear extracts

  • Analyze precipitates for TSA-sensitive histone deacetylase activity using a fluorescent HDAC assay

  • Include isotype-matched immunoglobulin and HDAC inhibitor (TSA) controls

• Protein complex analysis:

  • Perform gel filtration chromatography of nuclear extracts

  • Analyze fractions by Western blot for AJUBA, HDAC1, HDAC2, and HDAC3

  • Identify high molecular weight complexes containing both AJUBA and HDACs

• Chromatin-associated studies:

  • Conduct ChIP assays to identify genomic regions where AJUBA and HDACs co-localize

  • Perform sequential ChIP (ChIP-reChIP) to confirm co-occupancy

  • Quantify histone acetylation changes at specific loci upon AJUBA manipulation

• Functional assessment:

  • Utilize luciferase reporter assays with promoters of potential target genes

  • Compare wild-type AJUBA with mutants lacking HDAC interaction domains

  • Measure target gene expression changes with and without HDAC inhibitors

Published studies have established that AJUBA functions as an HDAC-dependent corepressor for specific target genes, including those regulated by Gfi1 . The LIM domains of AJUBA are particularly important for these interactions.

Why might I observe multiple bands when detecting AJUBA by Western blot?

Multiple bands in AJUBA Western blots may occur for several reasons:

• Expected pattern: AJUBA's primary band appears at 55-60 kDa , with a secondary uncharacterized band sometimes observed at approximately 30 kDa

• Potential causes of additional bands:

  • Post-translational modifications (phosphorylation)

  • Alternative splicing variants

  • Proteolytic cleavage during sample preparation

  • Cross-reactivity with related LIM domain proteins

  • Antibody specificity issues

• Validation approaches:

  • Compare patterns with validated positive controls (HeLa, HepG2, PC-3, A549, RKO cells)

  • Test multiple antibodies targeting different AJUBA epitopes

  • Perform siRNA knockdown or CRISPR knockout of AJUBA to identify specific bands

  • Treat samples with phosphatase to identify phosphorylated forms

• Technical considerations:

  • Ensure complete protease inhibition during sample preparation

  • Optimize SDS-PAGE conditions for better resolution

  • Consider using gradient gels for improved separation

How can I validate the specificity of my AJUBA antibody in biological samples?

To validate AJUBA antibody specificity:

• Genetic manipulation validation:

  • Perform siRNA-mediated knockdown or CRISPR/Cas9 knockout of AJUBA

  • Western blot analysis should show significantly reduced or absent signal with specific antibodies

  • This approach has been successfully used in published AJUBA studies

• Recombinant protein controls:

  • Test antibody against purified recombinant AJUBA protein

  • Include negative controls of related LIM domain proteins to assess cross-reactivity

• Multiple antibody validation:

  • Compare results using antibodies targeting different AJUBA epitopes:

    • N-terminal region (Met1-Arg121)

    • Mid-region (aa 200-350)

    • C-terminal LIM domains

• Immunoprecipitation-Western blot:

  • Immunoprecipitate with one AJUBA antibody

  • Detect with a different AJUBA antibody recognizing a distinct epitope

• Peptide competition:

  • Pre-incubate antibody with excess immunizing peptide

  • Specific signals should be blocked in subsequent immunodetection

• Tissue/cell expression pattern:

  • Compare observed expression patterns with published data

  • AJUBA has been detected in various tissues including skin, thyroid, small intestine, and testis

• Functional validation:

  • Confirm biological activities associated with AJUBA

  • For example, co-immunoprecipitation with known interacting partners (p53, MDM2, HDACs)

What technical considerations are important when using AJUBA Antibody, HRP conjugated in co-immunoprecipitation studies?

When using HRP-conjugated AJUBA antibodies in co-immunoprecipitation studies:

• Cross-linking considerations:

  • HRP conjugation may affect antibody binding properties

  • Consider using a non-conjugated AJUBA antibody for immunoprecipitation

  • For detection, either:

    • Use an HRP-conjugated secondary antibody against the primary IP antibody

    • Use a different HRP-conjugated AJUBA antibody that recognizes a distinct epitope

• Heavy/light chain interference:

  • HRP-conjugated antibodies used for both IP and detection will show heavy/light chain bands

  • Solutions include:

    • Using TrueBlot® or similar secondary antibodies that preferentially detect native immunoglobulins

    • Employing antibodies raised in different species for IP and detection

    • Using conjugated protein A/G for detection instead of secondary antibodies

• Antibody elution strategies:

  • For sequential co-IP studies of complexes like AJUBA-p53-MDM2:

    • Consider using epitope tag systems (FLAG, HA, Myc) for clean elution

    • Elute first-round complexes using specific peptides (e.g., 3X FLAG peptide)

    • Perform second-round IP with antibodies against the next protein in the complex

• Buffer optimization:

  • For detecting transient or weak interactions:

    • Use gentler lysis conditions with lower detergent concentrations

    • Include protein crosslinkers like DSP or formaldehyde before lysis

    • Consider adding phosphatase inhibitors to preserve phosphorylation-dependent interactions

Published studies have successfully demonstrated AJUBA's interactions with multiple partners using carefully optimized co-immunoprecipitation approaches .

How is AJUBA being investigated in cancer therapeutic development?

Recent discoveries about AJUBA's role in cancer progression have opened several therapeutic avenues:

• Targeting AJUBA-p53 interaction:

  • AJUBA overexpression reduces cancer cell sensitivity to chemotherapeutic drugs

  • Inhibiting AJUBA-p53-MDM2 complex formation could potentially restore chemosensitivity

  • Structural studies of AJUBA's C-terminal LIM domain interaction with p53's DNA-binding domain provide targets for small molecule development

• AJUBA as a biomarker:

  • High AJUBA expression correlates with poorer survival in colorectal cancer patients

  • AJUBA expression could potentially serve as a predictive biomarker for chemotherapy response

  • CdGAP mRNA levels inversely correlate with E-cadherin expression, with AJUBA as a mediator

• Exploiting the AJUBA-chemotherapy feedback loop:

  • Chemotherapeutic drugs induce AJUBA expression in a p53-dependent manner

  • This creates a negative feedback loop that can limit therapeutic efficacy

  • Combination therapies targeting both cancer cells and this feedback mechanism may improve outcomes

• AJUBA in tumor-stroma interactions:

  • AJUBA functions as a receptor mediating internalization of tumor-secreted GRP78 into macrophages

  • This reveals a pro-survival strategy where tumor cells educate stromal cells

  • Blocking this pathway could potentially impair tumor-stroma communication

These research directions highlight AJUBA's potential as both a therapeutic target and a biomarker in cancer treatment strategies.