PBX3 Antibody

What is PBX3 and why is it important in research?

PBX3 (Pre-B-cell leukemia homeobox 3) is a transcription factor with a calculated molecular weight of approximately 47 kDa that plays crucial roles in DNA-binding, transcriptional regulation, and development . Recent studies have identified PBX3 as a multifunctional oncogene involved in tumor growth, invasion, and metastasis in various cancers, particularly in papillary thyroid carcinoma (PTC) .

PBX3 enables several key biological functions:

• DNA-binding transcription factor activity

• RNA polymerase II-specific binding

• Regulation of transcription by RNA polymerase II

• Animal organ and neuron development

The protein is primarily located in the nucleus as part of chromatin complexes, where it interacts with Hox proteins to increase DNA binding specificity and regulate target gene expression . This makes PBX3 antibodies essential tools for studying cancer progression, transcriptional regulation, and developmental processes.

What are the primary applications of PBX3 antibodies in research?

PBX3 antibodies serve as versatile tools across multiple research applications, enabling detailed investigation of this protein's expression, localization, and interactions.

These techniques have been essential in discovering PBX3's upregulation in cancer and its correlation with clinical parameters . For instance, immunohistochemistry staining has been used to analyze PBX3 expression in PTC tissues, revealing significant associations with tumor size, lymphatic metastasis, and TMN stage .

How should PBX3 antibodies be validated for experimental use?

Antibody validation is critical for ensuring reliable and reproducible research results. For PBX3 antibodies, comprehensive validation should include:

Essential Validation Methods

• Positive and Negative Controls

  • Use cell lines with known PBX3 expression (e.g., TPC-1, SW579, BCPAP for PTC cell lines)

  • Include PBX3 knockdown controls (using shRNA-PBX3)

  • Compare expression between cancer and normal cell lines (e.g., PTC cell lines vs. NO3-1 normal thyroid cells)

• Multiple Detection Methods

  • Verify findings across different techniques (WB, IHC, IF)

  • Confirm appropriate molecular weight (~47 kDa) in Western blot analyses

• Genetic Approaches

  • Test antibody in PBX3 knockdown and overexpression systems

  • In studies, PBX3 expression was manipulated using lentiviral vectors expressing shRNA or overexpression constructs

Validation Data Example

Research has demonstrated PBX3 validation through multiple approaches:

• qRT-PCR analysis in 20 pairs of PTC samples versus adjacent normal tissue

• Western blot confirmation in clinical samples

• Expression comparison across five PTC cell lines versus normal thyroid cells

• Immunohistochemistry correlation with clinical parameters

These multiple validation approaches confirmed that PBX3 is significantly upregulated in PTC tissues compared to adjacent normal tissues, providing confidence in antibody specificity and experimental results .

Western Blot Protocol for PBX3 Detection

• Sample Preparation

  • Lyse cells in appropriate buffer with protease inhibitors

  • Load 20-50 μg of total protein per lane

• SDS-PAGE and Transfer

  • Separate proteins on 10-12% SDS-PAGE gel

  • Transfer to membrane (PVDF or nitrocellulose)

• Antibody Incubation

  • Block membrane with 5% non-fat milk or BSA

  • Incubate with PBX3 antibody (1:500-1:1000) overnight at 4°C

  • Wash thoroughly with TBST

  • Incubate with HRP-conjugated secondary antibody for 1-1.5 hours

• Detection

  • Apply ECL substrate and visualize

  • Expected band: ~47 kDa for PBX3

Immunohistochemistry Protocol

• Tissue Preparation

  • Fix tissues in formalin and embed in paraffin

  • Section at 4-6 μm thickness

  • Deparaffinize and rehydrate

• Antigen Retrieval and Antibody Incubation

  • Perform heat-induced epitope retrieval

  • Block endogenous peroxidase and non-specific binding

  • Incubate with PBX3 antibody (1:20-1:200) overnight at 4°C

  • Apply HRP-conjugated secondary antibody

• Detection

  • Develop with DAB substrate

  • Counterstain with hematoxylin

This approach has been successfully used to analyze PBX3 expression in tumor tissues and correlate it with clinical parameters such as tumor size, lymphatic metastasis, and TMN stage .

How does PBX3 expression correlate with cancer progression?

Research has established strong correlations between PBX3 expression and cancer progression, particularly in papillary thyroid carcinoma (PTC):

Functional Impact

Experimental manipulation of PBX3 expression has revealed its oncogenic functions:

• PBX3 Overexpression Promotes:

  • Cell proliferation

  • Migration and invasion

  • G0/G1 to S phase cell cycle transition

  • Angiogenesis

• PBX3 Knockdown Inhibits:

  • Cell cycle progression (induces G0/G1 arrest)

  • Tumor growth in vitro and in vivo

  • Angiogenesis (reduced VEGF levels and endothelial tube formation)

These findings suggest PBX3 could serve as both a prognostic biomarker and potential therapeutic target for cancer treatment.

Signaling Pathway Activation

PBX3 promotes PTC progression through activation of the AT1R/VEGFR2 pathway:

• Activates VEGFR2 phosphorylation

• Triggers downstream signaling through ERK1/2, AKT, and Src

• Overexpression of AT1R and treatment with VEGFA can reverse the effects of PBX3 knockdown

Cell Cycle Regulation

PBX3 accelerates G0/G1 to S phase transition:

• Flow cytometry analysis shows PBX3 knockdown increases cell proportion in G0/G1 phase

• PBX3 overexpression decreases G0/G1 phase cells and increases S phase cells

• Affects expression of cell cycle regulators (cyclins D1 and A)

Angiogenesis Promotion

PBX3 enhances tumor angiogenesis:

• Knockdown of PBX3 decreases expression of endothelial vascular marker CD31

• Reduces cell proliferation marker Ki-67 in tumor tissues

• PBX3-knockdown tumor-conditioned medium shows lower levels of VEGF

• Inhibits HUVEC tube formation and CAM angiogenesis

These mechanistic insights provide potential targets for developing therapeutics against PBX3-driven cancer progression.

What is known about PBX3's DNA binding properties and transcriptional activities?

PBX3 exhibits unique DNA binding characteristics that differentiate it from other PBX family members:

Interaction with Hox Proteins

• Forms heterodimeric complexes with Antennapedia family members (Hoxc-6, Hoxb-7, Hoxb-8)

• These complexes select the same binding site (TGATTTAT)

• The binding specificity of Hox proteins for optimal binding sites is increased in the presence of PBX proteins

Transcriptional Regulation

PBX3 is involved in regulating genes associated with:

• Cell cycle progression

• Angiogenesis

• Cell migration and invasion

• Development processes

These DNA binding properties highlight PBX3's role as a sophisticated transcriptional regulator capable of fine-tuning gene expression through diverse protein-protein and protein-DNA interactions.

What experimental approaches can be used to study PBX3 interactions with other proteins?

Several complementary techniques can be employed to investigate PBX3's protein-protein interactions:

Co-Immunoprecipitation (Co-IP)

• Use anti-PBX3 antibody to pull down PBX3 complexes

• Analyze by Western blot with antibodies against suspected binding partners

• Recommended antibody amount: 0.5-4.0 μg for 1-3 mg protein lysate

Chromatin Immunoprecipitation (ChIP)

• Identify genomic binding sites and co-factors

• Crosslink proteins to DNA before immunoprecipitation

• Analyze by qPCR or sequencing (ChIP-seq)

• Critical for understanding PBX3's role in transcriptional regulation

In Vitro Binding Assays

Researchers have used binding site selection strategies to determine optimal binding sites for PBX proteins alone and as heterodimeric partners with Hox gene products. These experiments revealed that:

• PBX3 can bind as a monomer or homodimer to specific DNA sequences

• When complexed with Hox proteins, PBX3 binds to distinct consensus sequences

• Binding affinities can be measured and compared across different protein complexes

These approaches allow for comprehensive characterization of PBX3's interaction network, providing insights into its role in normal development and disease processes.

Western Blot Troubleshooting

Immunohistochemistry/Immunofluorescence Troubleshooting

Special Considerations for PBX3

• Nuclear localization requires good permeabilization for IF/ICC

• For co-IP experiments, use buffers that preserve nuclear protein interactions

• When studying PBX3-Hox protein interactions, consider the specific DNA binding preferences of the complex

Careful optimization and validation of experimental conditions are essential for generating reliable data on PBX3 expression and function.

What are the best practices for storing and handling PBX3 antibodies?

Proper storage and handling of PBX3 antibodies are critical for maintaining their functionality and experimental reproducibility:

Storage Conditions

Handling Recommendations

• Avoid repeated freeze-thaw cycles that can degrade antibody quality

• Aliquot antibodies before freezing to minimize freeze-thaw cycles

• Allow antibodies to reach room temperature before opening vials

• Store with stabilizers (typically provided as 500 μg/ml antibody with PBS, 0.02% NaN₃, 1 mg BSA, and 50% glycerol)

• Follow manufacturer's recommendations for reconstitution if applicable

• Centrifuge antibody briefly before use to collect solution at the bottom of the vial

Following these storage and handling guidelines will help ensure consistent antibody performance across experiments and maximize the useful life of PBX3 antibodies.