IKZF3 Antibody

What is IKZF3 and why is it an important research target?

IKZF3 (Ikaros family zinc finger protein 3), also known as Aiolos or ZNFN1A3, is a transcription factor that plays crucial roles in lymphocyte differentiation. It is particularly important in regulating B-cell differentiation, proliferation, and maturation to an effector state. Additionally, IKZF3 is involved in regulating BCL2 expression and controlling apoptosis in T-cells in an IL-2-dependent manner . Research targeting IKZF3 is valuable for understanding immune cell development, function, and dysregulation in various disease states, particularly hematological malignancies.

What are the primary applications for IKZF3 antibodies in research?

IKZF3 antibodies have been validated for multiple research applications including:

How should I optimize flow cytometry protocols for IKZF3 detection in human lymphocytes?

For optimal detection of IKZF3 in human lymphocytes by flow cytometry:

• Sample preparation: Isolate PBMCs using ficoll-hypaque density gradient centrifugation (if using frozen samples, thaw cells in prewarmed complete medium) .

• Surface marker staining: First stain for surface markers (e.g., CD19 for B-cells) using appropriate fluorochrome-conjugated antibodies.

• Fixation and permeabilization: Use a dedicated fixation and permeabilization buffer system such as FlowX FoxP3 Fixation & Permeabilization Buffer Kit to enable intracellular staining .

• IKZF3 staining: Incubate with the primary anti-IKZF3 antibody at the optimal dilution (determined through titration).

• Secondary antibody: If using an unconjugated primary antibody, apply a fluorophore-conjugated secondary antibody (e.g., Phycoerythrin-conjugated Anti-Mouse IgG) .

• Controls: Include isotype controls (e.g., Mouse IgG2B Flow Cytometry Isotype Control for mouse-derived anti-IKZF3 antibodies) and both positive and negative cellular controls .

What are the critical considerations when performing Western blot analysis of IKZF3?

For successful Western blot detection of IKZF3:

• Lysate preparation: For optimal results, use freshly prepared lysates from cells known to express IKZF3 (e.g., Raji, Ramos, or Jurkat cell lines) .

• Protein loading: Load 20 μg of total protein per lane for cell line lysates; higher amounts may be needed for tissue samples .

• Antibody dilution: Use high-quality antibodies at appropriate dilutions (e.g., 1/20000 dilution for the EPR9342(B) clone) .

• Detection system: Use a sensitive detection system such as peroxidase-conjugated secondary antibodies at 1/1000 dilution .

• Band interpretation: The predicted molecular weight for IKZF3 is 58 kDa, but be aware of potential isoforms that may appear as additional bands .

How can I assess the functional implications of IKZF3 expression levels in T-cells?

To investigate the functional consequences of varying IKZF3 expression in T-cells:

What approaches can be used to study the influence of the tumor microenvironment on IKZF3 expression?

To investigate how the tumor microenvironment affects IKZF3 expression:

• Serum-mediated effects: Isolate CD3+ T-cells from healthy donors and culture them in medium containing 10% bone marrow (BM) serum from either MGUS patients (as control) or newly diagnosed MM stage III patients for 48 hours. Then analyze IKZF3 expression by flow cytometry .

• Gene expression analysis: Complement protein-level assessments with RT-PCR analysis of IKZF3 gene expression under different culture conditions .

• Comparative analysis: Compare IKZF3 expression in T-cells from patients with different disease stages (e.g., MM stage I vs. MM stage III) to assess disease progression effects .

• Interpretation challenges: Research has shown that IKZF3 expression in T-cells is not significantly affected by soluble factors in the tumor microenvironment, suggesting intrinsic rather than extrinsic regulation .

What is the prognostic and predictive value of IKZF3 expression in multiple myeloma patients?

IKZF3 expression levels in T-cells have significant prognostic implications in multiple myeloma:

How does IKZF3 expression affect T-cell response to immunomodulatory drugs?

The relationship between IKZF3 expression and response to immunomodulatory drugs presents an interesting paradox:

• Baseline T-cell functionality: High IKZF3 expression in T-cells is associated with impaired antigen-specific T-cell responses before treatment, suggesting immunosuppressive effects .

• Drug response enhancement: Despite baseline immunodeficiency features, patients with high IKZF3 expression show significantly enhanced IL-2 production in response to lenalidomide treatment compared to those with low IKZF3 expression .

• Mechanistic hypothesis: This paradoxical effect may be explained by the higher potential for improvement in T-cell function when immunomodulatory drugs target cells with elevated IKZF3 levels. IKZF3 might disrupt the IL-2 autocrine loop in T-cells, which immunomodulatory drugs can partially restore .

• Experimental validation: When testing this hypothesis, incubate isolated CD3+ T-cells from IKZF3-high and IKZF3-low MM patients with anti-CD3/CD28 activation beads and 10 μM lenalidomide or DMSO control for 24 hours. Measure IL-2 concentrations in supernatants by ELISA .

How can IKZF3 antibodies be used to investigate the relationship between IKZF3 and IL-10 expression in CD4+ T cells?

Recent research has indicated an association between IKZF3 (Aiolos) and anti-inflammatory IL-10 expression in CD4+ T cells, which is crucial for immune homeostasis . To investigate this relationship:

• Co-expression analysis: Use multi-parameter flow cytometry with antibodies against IKZF3 and IL-10 to analyze co-expression patterns in different CD4+ T cell subsets.

• Functional studies: Implement IKZF3 knockdown or overexpression experiments in CD4+ T cells, followed by analysis of IL-10 production after appropriate stimulation.

• Mechanistic investigations: Explore whether IKZF3 directly regulates IL-10 transcription through chromatin immunoprecipitation (ChIP) assays targeting the IL-10 promoter region.

• Correlation with clinical outcomes: Analyze the relationship between IKZF3 expression, IL-10 production, and clinical parameters in autoimmune diseases or inflammatory conditions.

What are the methodological approaches for studying IKZF3 degradation induced by immunomodulatory drugs?

To investigate drug-induced IKZF3 degradation mechanisms:

• Protein degradation assays: Treat relevant cell types with immunomodulatory drugs at various concentrations and time points, then analyze IKZF3 protein levels by Western blot.

• Proteasome inhibition: Include proteasome inhibitors (e.g., MG132) in parallel experiments to determine if degradation occurs through the ubiquitin-proteasome pathway.

• Ubiquitination analysis: Perform immunoprecipitation of IKZF3 followed by Western blot analysis for ubiquitin to assess drug-induced changes in ubiquitination status.

• Molecular imaging: Use fluorescently tagged IKZF3 constructs in live-cell imaging experiments to monitor protein degradation dynamics in real-time.