HNRNPAB Antibody, FITC conjugated

What is the biological function of HNRNPAB in normal and cancer cells?

HNRNPAB is an RNA binding protein primarily expressed in the nucleus that participates in multiple RNA processing functions including mRNA selective splicing, mRNA stabilization, and gene transcription regulation . In normal tissues, HNRNPAB contributes to basic RNA metabolism, but in cancer contexts, it becomes significantly overexpressed and contributes to malignant phenotypes.

Research findings indicate HNRNPAB is involved in:

• Epithelial-mesenchymal transition (EMT) promotion through regulating transcription factors such as SNAI1

• Cell cycle progression, particularly at G1 phase transition

• Maintenance of cancer stem cell (CSC) properties, particularly in colorectal cancer

• Regulation of tumorigenesis-related gene expression networks

The protein demonstrates nuclear localization in most cancer cells, where it exerts its RNA binding and processing functions to affect downstream oncogenic pathways .

Which cancer types show significant HNRNPAB dysregulation?

HNRNPAB expression has been studied across multiple cancer types with consistent findings of overexpression compared to corresponding normal tissues:

Database analysis across multiple tumor types confirms HNRNPAB's value as both a prognostic marker and potential therapeutic target .

What are the optimal protocols for HNRNPAB detection in immunofluorescence studies?

For effective HNRNPAB detection using FITC-conjugated antibodies, researchers should consider:

Cell Fixation and Permeabilization:

• Paraformaldehyde fixation (4%) for 15-20 minutes at room temperature preserves cellular architecture

• Permeabilization with 0.2% Triton X-100 in PBS for 10 minutes enables antibody access to nuclear targets

• Blocking with 3-5% BSA or normal serum for 1 hour reduces non-specific binding

Antibody Dilution and Incubation:
Based on published protocols, optimal dilutions typically range from 1:100 to 1:500, with incubation at 4°C overnight for primary antibody steps. For FITC-conjugated antibodies, protection from light during all steps is critical to prevent photobleaching .

Nuclear Counterstaining:
Since HNRNPAB is primarily nuclear, counterstaining with DAPI (1:1000) provides spatial context for evaluating specific nuclear localization patterns observed in cancer cells .

Research has confirmed that HNRNPAB predominantly localizes to the nucleus in NSCLC cells, which should be verified through co-localization with nuclear markers in any experimental system .

How can HNRNPAB antibodies be validated for experimental specificity?

Rigorous validation is essential when studying HNRNPAB expression:

Controls for Immunostaining Validation:

• Positive control: Use cell lines with known high HNRNPAB expression (NCI-H292, PC-9 for NSCLC studies)

• Negative control: HNRNPAB-knockdown cells created using validated shRNA constructs

• Peptide competition assay: Pre-incubation of antibody with purified HNRNPAB peptide should abolish signal

Western Blot Validation:
The antibody should detect a band at approximately the expected molecular weight for HNRNPAB (approximately 36 kDa). Published research employs rabbit polyclonal antibodies against HNRNPAB (e.g., A17497, 1:2,000 dilution, ABclonal) .

Knockdown Validation:
HNRNPAB-targeting shRNA constructs have been validated in multiple studies. Researchers should expect at least 70% reduction in protein levels after stable knockdown, which can be accomplished using lentiviral vectors containing constructs such as those described in published research .

How can researchers design functional studies to investigate HNRNPAB's role in cancer progression?

Based on published methodologies, robust functional analysis requires:

Knockdown Studies:

• Design target-specific shRNAs (researchers have successfully used psi-LVRU6GP vector systems)

• Validate knockdown efficiency via both RT-qPCR and Western blot

• For stable lines, puromycin selection (2.0 μg/ml) has proven effective after 72 hours of lentiviral infection

Functional Assays to Assess:

• Proliferation: MTT or similar viability assays comparing knockdown and control cells

• Migration: Wound healing or transwell migration assays

• Invasion: Matrigel-coated transwell chambers

• EMT: Assessment of markers including E-cadherin, N-cadherin, Vimentin, ZEB1, and SNAI1

Cell Cycle Analysis:
Flow cytometry with propidium iodide staining can reveal HNRNPAB's effect on cell cycle progression, with knockdown studies showing G1 phase arrest in NSCLC cells .

What methodological approaches enable investigation of HNRNPAB in cancer stem cell biology?

Research indicates HNRNPAB influences cancer stem cell properties, particularly in colorectal cancer . Key methodological considerations include:

Isolation of Cancer Stem Cell Populations:

• Flow cytometry sorting using established CSC markers (CD44+/CD24-, ALDH+)

• Sphere formation assays under low-attachment conditions

• Serial dilution tumorigenic assays in animal models

Chemosensitivity Testing:

• Treatment with standard chemotherapeutics (e.g., 5-FU, oxaliplatin) in HNRNPAB-modulated cells

• Combination studies with CSC pathway inhibitors

• Analysis of apoptotic markers following treatment

Gene Expression Analysis:

• RT-qPCR panel analysis of stemness markers

• Pathway analysis focusing on Wnt/β-catenin, Notch, and Hedgehog signaling

• Assessment of correlation between HNRNPAB levels and established stemness signatures

Research has demonstrated that knockdown of HNRNPAB enhances chemosensitivity of colorectal cancer stem cells, suggesting therapeutic implications .

What methods are most effective for studying HNRNPAB's RNA binding targets?

As an RNA-binding protein, HNRNPAB's function depends on its interactions with specific RNA targets:

RNA Immunoprecipitation (RIP):

• Use validated HNRNPAB antibodies for immunoprecipitation

• Cross-link RNA-protein complexes (UV or formaldehyde)

• Analyze bound RNAs by RT-qPCR or RNA sequencing

Crosslinking and Immunoprecipitation (CLIP):

• Allows precise mapping of binding sites

• Ultraviolet crosslinking provides higher resolution of binding locations

• Combined with high-throughput sequencing for genome-wide target identification

Functional Validation:

• Luciferase reporter constructs containing predicted binding sites

• Site-directed mutagenesis of binding motifs

• RNA electrophoretic mobility shift assays (REMSA)

Research has identified G-rich elements as binding sites for related hnRNP family members (hnRNP H and F), which act as splicing enhancers when located downstream of target exons . Similar approaches could be applied to HNRNPAB studies.

How can researchers investigate the relationship between HNRNPAB and EMT mechanisms?

HNRNPAB has been implicated in promoting EMT in multiple cancer types:

EMT Marker Analysis:
After HNRNPAB knockdown or overexpression, researchers should assess:

• Epithelial markers: E-cadherin

• Mesenchymal markers: N-cadherin, Vimentin

• EMT transcription factors: SNAI1, ZEB1

Mechanistic Studies:

• Chromatin immunoprecipitation (ChIP) to assess direct regulation of EMT genes

• Analysis of HNRNPAB binding to EMT regulator mRNAs

• Rescue experiments by reintroducing EMT factors in HNRNPAB-depleted cells

Pathway Integration:
Research indicates HNRNPAB knockdown significantly affects expression of genes associated with tumorigenesis, suggesting broader pathway effects beyond direct targets .

What methodologies enable effective correlation of HNRNPAB expression with patient outcomes?

Translational studies require robust approaches to link HNRNPAB with patient data:

Database Analysis Methods:

• The Cancer Genome Atlas (TCGA) data mining (used for 585 LUAD and 550 LUSC cases)

• Kaplan-Meier survival analysis using median expression as cutoff

• Cox proportional hazard models for multivariate analysis

• Chi-square tests for correlations with clinicopathological parameters

Tissue Microarray Analysis:

• Immunohistochemical staining of patient tissue arrays

• Scoring systems based on staining intensity and percentage of positive cells

• Correlation with clinicopathological features and survival data

How can HNRNPAB analysis be integrated into broader cancer biomarker panels?

HNRNPAB exists within complex regulatory networks, requiring integrated analysis approaches:

Multi-marker Panel Development:

• Combine with other hnRNP family members (researchers have identified HNRNPU and SYNCRIP as core molecular genes in invasive breast carcinoma)

• Include complementary markers reflecting EMT status, proliferation, and stemness

• Develop multiplexed immunofluorescence or flow cytometry panels

Bioinformatic Integration:

• Gene set enrichment analysis (researchers have found negative correlation between hnRNP H and Myc hallmarks)

• Protein-protein interaction networks

• Co-expression analysis to identify functional associations

Validation Strategies:

• Cross-validation across independent cohorts

• Comparison with established clinical biomarkers

• Integration with molecular subtypes

Research on the broader hnRNP family suggests specific members could serve as therapeutic targets and prognostic biomarkers, with HNRNPU and SYNCRIP identified as core molecular genes requiring further investigation in breast carcinoma .