MYBL2 Antibody

What is MYBL2 and why is it important as a research target?

MYBL2 (MYB proto-oncogene like 2), also known as B-MYB, is a transcription factor involved in cell cycle regulation, DNA replication, and cell survival. It belongs to the MYB family of proteins and plays crucial roles in regulating biological processes including:

• Cell cycle progression, particularly at the G2/M checkpoint

• DNA double-strand break repair mechanisms

• Tumor proliferation and phenotypic plasticity

• Immune response modulation in the tumor microenvironment

Recent studies have identified MYBL2 as a significant prognostic biomarker in various cancers, including pancreatic, prostate, and ovarian cancers. Its expression is often dysregulated in tumor tissues compared to normal tissues, making it an important target for cancer research .

When selecting MYBL2 as a research target, researchers should consider:

• The specific cellular processes being investigated

• Cancer type and stage relevance

• Potential correlation with treatment responses

• Association with specific signaling pathways (e.g., PI3K/AKT, p53)

What experimental techniques are commonly employed for MYBL2 detection in research?

MYBL2 detection employs several complementary techniques that provide different types of information:

For comprehensive MYBL2 analysis, researchers should employ multiple techniques to validate findings. For instance, when studying MYBL2's role in pancreatic cancer, researchers analyzed both mRNA expression using RNA-sequencing and protein expression via immunohistochemistry to establish MYBL2 as a prognostic biomarker .

How should MYBL2 antibodies be validated before experimental use?

Proper validation of MYBL2 antibodies is critical for ensuring reliable research outcomes. A systematic validation approach should include:

• Specificity testing:

  • Western blot analysis with positive controls (e.g., cells transfected with MYBL2 expression vectors)

  • Comparison with negative controls (e.g., cells with MYBL2 knockdown)

  • Testing in multiple cell lines with known MYBL2 expression levels

• Cross-reactivity assessment:

  • Testing on tissue samples from different species based on the antibody's claimed reactivity

  • Evaluating potential cross-reactivity with other MYB family members (MYB, MYBL1)

• Functional validation:

  • Correlation of staining patterns with functional readouts

  • Verification that antibody detection corresponds with phenotypic effects

As demonstrated in antibody validation studies, researchers have used 293 HEK cells transfected with human B-Myb cDNA alongside negative controls (pcDNA3 vector) to confirm antibody specificity through Western blot analysis . In vitro translation in rabbit reticulocyte lysate provides another validation approach to confirm the molecular weight and specificity of the detected band.

What considerations are important when using MYBL2 antibodies for cancer prognosis studies?

When designing experiments to evaluate MYBL2 as a prognostic marker, researchers should implement the following methodological approaches:

• Sample selection and preparation:

  • Include matched tumor and adjacent normal tissues when possible

  • Standardize tissue processing protocols (fixation times, antigen retrieval methods)

  • Collect comprehensive clinical follow-up data including survival outcomes

• Scoring and quantification:

  • Establish clear scoring systems (e.g., H-score, percentage positive cells)

  • Use digital pathology for objective quantification

  • Implement blinded evaluation by multiple observers

• Statistical analysis:

  • Perform Kaplan-Meier survival analysis with log-rank tests

  • Conduct univariate and multivariate Cox proportional hazards regression

  • Calculate ROC curves to determine optimal cutoff values

In pancreatic cancer studies, researchers have demonstrated that MYBL2 expression correlates significantly with cancer grade and stage through univariate and multivariate factor analyses. Statistical significance was established using Wilcox tests with significance levels clearly indicated (*p < 0.05, **p < 0.01, ***p < 0.001) .

To enhance reproducibility, researchers should also document antibody specifications including clone, dilution, incubation conditions, and detection systems employed.

How can researchers effectively use MYBL2 antibodies to study cell cycle regulation mechanisms?

To investigate MYBL2's role in cell cycle regulation, researchers can employ these methodological approaches:

• Cell cycle synchronization and analysis:

  • Synchronize cells at specific cell cycle phases using methods like double thymidine block or nocodazole treatment

  • Perform flow cytometry with MYBL2 antibodies and DNA content staining

  • Analyze MYBL2 expression levels across different cell cycle phases

• Knockdown and overexpression studies:

  • Use siRNA-mediated MYBL2 silencing or MYBL2 overexpression vectors

  • Measure cell cycle distribution using flow cytometry

  • Document phenotypic changes including proliferation rates and cell morphology

• Downstream target identification:

  • Combine MYBL2 antibodies with ChIP-seq to identify binding sites

  • Perform RNA-seq after MYBL2 modulation to identify regulated genes

  • Validate key targets using qRT-PCR and Western blotting

Research has demonstrated that MYBL2 silencing in gastric cancer cell lines significantly increased the percentage of cells in G2/M phase, while MYBL2 overexpression increased the proportion of cells in S phase. These findings were quantified using flow cytometry, establishing MYBL2's role in modulating G2/S cell phase transition .

What protocols are recommended for using MYBL2 antibodies in Western blot analysis?

For optimal Western blot results when detecting MYBL2, researchers should follow this methodological protocol:

• Sample preparation:

  • Lyse cells in RIPA buffer supplemented with protease and phosphatase inhibitors

  • Quantify protein using BCA or Bradford assay

  • Denature samples at 95°C for 5 minutes in loading buffer with reducing agent

• Gel electrophoresis and transfer:

  • Load 20-50 μg protein per lane on 8-10% SDS-PAGE gels (MYBL2 is approximately 78.8 kDa)

  • Use pre-stained molecular weight markers

  • Transfer to PVDF membrane at 100V for 90 minutes in cold transfer buffer

• Antibody incubation:

  • Block membrane in 5% non-fat milk in TBST for 1 hour at room temperature

  • Incubate with primary MYBL2 antibody at manufacturer's recommended dilution (typically 1:500-1:2000) overnight at 4°C

  • Wash 3x with TBST, then incubate with appropriate HRP-conjugated secondary antibody

• Detection and analysis:

  • Develop using enhanced chemiluminescence (ECL) substrate

  • Expose to X-ray film or capture using digital imaging system

  • Normalize MYBL2 signal to loading control (β-actin, GAPDH)

As demonstrated in published protocols, dilution of MYBL2 antibody to approximately 200 μg/ml with exposure times of around 10 minutes has been effective for detecting MYBL2 in transfected cell lysates . Including both positive controls (MYBL2-transfected cells) and negative controls is critical for confirming specificity.

How can MYBL2 antibodies be utilized to evaluate immune infiltration in cancer specimens?

Studying MYBL2's relationship with tumor immune infiltration requires methodical approaches:

• Multiplex immunohistochemistry setup:

  • Perform sequential staining using MYBL2 antibodies and immune cell markers

  • Include CD68 (macrophages), CD8 (cytotoxic T cells), and other relevant immune markers

  • Use multispectral imaging systems for co-localization analysis

• Quantitative spatial analysis:

  • Map tumor regions versus stromal compartments

  • Quantify immune cell density in proximity to MYBL2-positive cells

  • Analyze spatial relationships using digital pathology software

• Correlation with clinical parameters:

  • Assess association between MYBL2 expression and immune infiltration

  • Correlate findings with treatment response data

  • Perform survival analysis based on combined MYBL2 and immune profiles

Research has demonstrated significant positive correlation between MYBL2 expression and macrophage infiltration in cancer tissues. For example, immunohistochemical staining for CD68 in serial tumor tissue slices confirmed that high MYBL2 expression positively correlated with CD68+ cell numbers in ovarian cancer specimens . Similarly, computational analysis using tools like TIMER2.0 has enabled researchers to examine correlations between MYBL2 expression and various immune cell populations in prostate cancer .

What role do MYBL2 antibodies play in investigating tumor mutational burden (TMB) and immunotherapy response prediction?

To investigate MYBL2's relationship with TMB and immunotherapy response, researchers should implement:

• Sequential analysis workflow:

  • Characterize MYBL2 expression using immunohistochemistry

  • Assess TMB through next-generation sequencing

  • Perform correlation analysis between MYBL2 levels and TMB scores

• Predictive model development:

  • Integrate MYBL2 expression data with TMB scores

  • Include clinical response data to immunotherapy (particularly PD-1/PD-L1 inhibitors)

  • Develop predictive algorithms using machine learning approaches

• Functional validation:

  • Test immunotherapy efficacy in models with varied MYBL2 expression

  • Monitor changes in immune cell composition and function

  • Assess cytokine profiles and immune activation markers

Research has identified a significant positive association between MYBL2 expression and tumor mutational burden (TMB) in pancreatic cancer, suggesting MYBL2 may enhance immunotherapy efficacy. This correlation was established using bioinformatics analysis of TCGA database data, demonstrating MYBL2's potential as a predictive biomarker for PD1 antibody treatment response .

How should researchers approach studying MYBL2's role in DNA repair mechanisms using antibody-based techniques?

To investigate MYBL2's function in DNA double-strand break (DSB) repair, implement these methodological approaches:

• DNA damage induction and repair kinetics:

  • Induce DSBs using ionizing radiation or radiomimetic drugs

  • Track γH2AX foci formation and resolution over time

  • Co-stain for MYBL2 and DSB repair proteins (e.g., RAD51, 53BP1)

• Chromatin immunoprecipitation (ChIP) analysis:

  • Perform ChIP using MYBL2 antibodies before and after DNA damage

  • Analyze recruitment to DNA damage sites

  • Identify damage-responsive target genes

• Functional repair assays:

  • Conduct comet assays to quantify DNA damage repair

  • Implement reporter assays for homologous recombination and non-homologous end joining

  • Measure telomere integrity in contexts of varied MYBL2 expression

Studies have demonstrated that MYBL2 haploinsufficiency in mice leads to defects in DSB repair induced by ionizing radiation in hematopoietic stem cells. This was characterized by unsustained phosphorylation of the ATM substrate KAP1 and telomere fragility. These findings established MYBL2 as a crucial regulator of DSB repair, suggesting MYBL2 expression levels as a potential biomarker to predict cellular response to genotoxic treatments .

What are common technical challenges when using MYBL2 antibodies and how can they be addressed?

Researchers frequently encounter these technical issues when working with MYBL2 antibodies:

For specific applications like immunohistochemistry, optimizing antigen retrieval methods is critical. Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) can significantly improve staining quality. Testing multiple dilutions (1:100, 1:200, 1:500) in pilot experiments will help identify optimal conditions for each antibody lot.

When working with challenging samples like formalin-fixed tissue, extended antibody incubation (overnight at 4°C) often yields better results than shorter incubations at room temperature.

How can researchers optimize MYBL2 antibody-based detection in samples with low MYBL2 expression?

For detecting low levels of MYBL2, implement these methodological enhancements:

• Signal amplification strategies:

  • Use tyramide signal amplification (TSA) systems for immunohistochemistry

  • Implement biotin-streptavidin amplification systems

  • Consider polymer-based detection methods with multiple HRP molecules

• Sample enrichment approaches:

  • Perform immunoprecipitation before Western blotting

  • Use cell fractionation to isolate nuclear proteins

  • Concentrate proteins using TCA precipitation or similar methods

• Advanced detection systems:

  • Employ highly sensitive chemiluminescent substrates for Western blots

  • Use digital imaging systems with adjustable exposure settings

  • Consider multiphoton microscopy for tissue samples with high autofluorescence

• Protocol adjustments:

  • Extend primary antibody incubation time (overnight at 4°C)

  • Decrease washing stringency slightly (reduce detergent concentration)

  • Use larger sample amounts where possible

These approaches have proven effective in detecting MYBL2 in samples with varied expression levels across multiple cancer types, enabling researchers to detect even subtle changes in MYBL2 expression that correlate with disease progression .

How can MYBL2 antibodies be employed to investigate the MYBL2-PI3K/AKT signaling axis?

To study MYBL2's interaction with the PI3K/AKT pathway, researchers should implement these methodological approaches:

• Pathway component analysis:

  • Perform Western blotting for MYBL2, p-PI3K, and p-AKT following MYBL2 modulation

  • Use co-immunoprecipitation to detect direct interactions between MYBL2 and pathway components

  • Apply proximity ligation assays to visualize protein-protein interactions in situ

• Pharmacological intervention studies:

  • Treat cells with PI3K/AKT inhibitors (e.g., MK2206) and assess MYBL2 expression

  • Combine MYBL2 overexpression with pathway inhibitors to assess rescue effects

  • Perform time-course experiments to determine signaling sequence

• Functional readouts:

  • Monitor cell proliferation, apoptosis, and cell cycle progression

  • Assess transcriptional activity using reporter assays

  • Evaluate phenotypic changes in 3D culture systems or in vivo models

Research has demonstrated that MYBL2 silencing significantly downregulates p-PI3K and p-AKT expression in gastric cancer cells, while MYBL2 overexpression upregulates these phosphorylated proteins. Importantly, AKT inhibitor (MK2206) treatment can reverse the proliferation effects induced by MYBL2 overexpression, confirming the functional relevance of this signaling axis .

What methodologies are recommended for investigating MYBL2's role in tumor-associated macrophage recruitment?

To study MYBL2's influence on tumor-associated macrophages (TAMs), researchers should employ:

• Macrophage recruitment assessment:

  • Use transwell migration assays with conditioned media from MYBL2-modulated cells

  • Perform immunohistochemistry for CD68+ macrophages in relation to MYBL2 expression

  • Implement live cell imaging to track macrophage migration in co-culture systems

• Mechanistic pathway analysis:

  • Analyze CCL2 expression using qRT-PCR and ELISA following MYBL2 modulation

  • Perform ChIP assays to determine if MYBL2 directly binds the CCL2 promoter

  • Test neutralizing antibodies against CCL2 to confirm specificity of effect

• Functional phenotyping:

  • Characterize macrophage polarization (M1/M2) through flow cytometry

  • Assess cytokine profiles in the tumor microenvironment

  • Evaluate therapeutic implications using PD-1 inhibitors in combination with MYBL2/CCL2 targeting

Research has identified a MYBL2-CCL2 axis in ovarian cancer, where tumor-derived MYBL2 transcriptionally activates CCL2, inducing TAM recruitment and M2-like polarization. This mechanism contributes to immune evasion and anti-PD-1 resistance. Notably, inhibition of CDK2 (a MYBL2 upstream kinase) using CVT-313 reprogrammed the tumor microenvironment and reduced resistance to immunotherapy .

How can researchers utilize MYBL2 antibodies to study its role in cancer phenotypic plasticity?

To investigate MYBL2's contribution to phenotypic plasticity in cancer, implement these methodological approaches:

• Multi-omics characterization:

  • Combine MYBL2 immunohistochemistry with transcriptomic profiling

  • Perform single-cell analysis to identify subpopulations with varying MYBL2 expression

  • Correlate MYBL2 levels with stemness and differentiation markers

• Lineage tracking experiments:

  • Develop reporter systems for MYBL2 expression in living cells

  • Track phenotypic changes over time following treatment challenges

  • Use lineage tracing in animal models to monitor cell fate transitions

• Functional manipulation studies:

  • Perform genetic inhibition of MYBL2 using CRISPR/Cas9 or shRNA

  • Assess changes in gene expression signatures for pluripotency and stemness

  • Evaluate in vivo tumor growth and metastatic potential

Research has demonstrated that genetic inhibition of Mybl2 in prostate cancer cell lines significantly decreased in vivo growth and cell fitness while repressing gene expression signatures involved in pluripotency and stemness. Since MYBL2 is not directly druggable, researchers developed an innovative approach using a MYBL2 gene signature to identify CDK2 as a potential therapeutic target. CDK2 inhibition was shown to phenocopy genetic loss of Mybl2, significantly decreasing in vivo tumor growth associated with DNA damage enrichment .

What emerging technologies can enhance MYBL2 antibody-based research in cancer studies?

Cutting-edge technologies that can advance MYBL2 research include:

• Spatial transcriptomics integration:

  • Combine MYBL2 immunohistochemistry with spatial transcriptomics

  • Map MYBL2 protein expression to transcriptional neighborhoods

  • Identify spatial relationships between MYBL2+ cells and specific microenvironmental niches

• Advanced imaging systems:

  • Implement imaging mass cytometry for highly multiplexed protein detection

  • Use light-sheet microscopy for 3D visualization of MYBL2 in organoids or tissues

  • Apply super-resolution microscopy to study MYBL2 nuclear organization

• Single-cell multi-omics:

  • Perform single-cell proteogenomics to correlate MYBL2 protein with transcriptional profiles

  • Apply CyTOF for simultaneous detection of MYBL2 and multiple signaling nodes

  • Develop computational approaches to integrate single-cell data across platforms

• Liquid biopsy applications:

  • Develop sensitive assays for MYBL2 detection in circulating tumor cells

  • Correlate MYBL2 expression with ctDNA profiles

  • Monitor treatment responses using serial liquid biopsies

These emerging technologies will enable more comprehensive understanding of MYBL2's role in cancer biology, potentially leading to improved prognostic tools and therapeutic strategies for cancers where MYBL2 drives disease progression .

What are the most promising research directions for MYBL2 antibody applications in precision oncology?

Based on current research findings, these directions show particular promise:

• Predictive biomarker development:

  • Standardized MYBL2 immunohistochemistry protocols for clinical implementation

  • Combined analysis of MYBL2 with TMB for immunotherapy response prediction

  • Integration of MYBL2 into multi-marker prognostic panels

• Therapeutic target identification:

  • Using MYBL2 antibodies to screen for synthetic lethal interactions

  • Identifying druggable downstream targets in the MYBL2 pathway

  • Developing antibody-drug conjugates targeting MYBL2-overexpressing cells

• Resistance mechanism characterization:

  • Studying MYBL2's role in therapy-induced phenotypic transitions

  • Monitoring MYBL2 dynamics during treatment to predict resistance

  • Targeting MYBL2-dependent pathways to overcome treatment resistance

Current evidence suggests MYBL2 can serve as a "double marker" for independent diagnosis and PD1 antibody response prediction in pancreatic cancer patients . Similarly, high MYBL2 activity identifies prostate cancers that may be responsive to CDK2 inhibition . The MYBL2-CCL2 axis presents another promising target to enhance immunotherapy efficacy in ovarian cancer .

By leveraging antibody-based detection of MYBL2 in these contexts, researchers may significantly advance personalized treatment strategies for multiple cancer types where MYBL2 drives disease progression.