CSTF2 Antibody

What is CSTF2 and why is it important in cancer research?

CSTF2 (Cleavage stimulation factor subunit 2) is a fundamental factor in the regulation of 3'-end cleavage and alternative polyadenylation (APA) of pre-mRNAs. Research has established that CSTF2 plays significant roles in multiple cancers. In hepatocellular carcinoma (HCC), CSTF2 is overexpressed and correlates with poor prognosis and high histological grades . It promotes tumor development through mechanisms including enhanced glycolysis via HK2 3'UTR shortening . In lung cancer, CSTF2 expression is associated with poor patient outcomes and is an independent prognostic factor . Additionally, CSTF2 has been identified as a key player in pancreatic ductal adenocarcinoma (PDAC), where it regulates mRNA N6-methyladenosine modification and influences cancer subtyping . The consistent finding of CSTF2 overexpression across multiple cancer types highlights its potential as both a prognostic biomarker and therapeutic target.

How do I select the appropriate CSTF2 antibody for my research?

When selecting a CSTF2 antibody, consider these critical factors:

• Application specificity: Determine if the antibody has been validated specifically for your intended application (WB, IHC, IF/ICC, flow cytometry). An antibody showing specificity in one application may not perform well in another .

• Species reactivity: Verify the antibody's reactivity with your species of interest. Available CSTF2 antibodies commonly react with human and mouse samples .

• Clonality: Consider whether a monoclonal or polyclonal antibody better suits your needs. Polyclonal antibodies may provide higher sensitivity but potentially lower specificity .

• Validation methods: Examine the validation data provided by manufacturers. Look for antibodies validated through multiple complementary methods such as knockout verification, siRNA knockdown, or overexpression systems .

• Epitope location: For specific applications, the location of the epitope (e.g., N-terminal vs. C-terminal) may be important. Some CSTF2 antibodies target the N-terminal region of the protein .

Review the antibody datasheets carefully for this information and consider consulting published literature where the antibody has been successfully used.

What are the optimal conditions for using CSTF2 antibodies in Western blotting?

For optimal Western blotting with CSTF2 antibodies, follow these methodological guidelines:

• Sample preparation:

  • Extract proteins from cells under native conditions using RIPA buffer supplemented with protease inhibitors

  • For tissues, particularly HCC or other cancer tissues, immediate freezing post-collection is crucial

• Dilution optimization:

  • Begin with manufacturer-recommended dilutions, typically 1:500 to 1:3000 for CSTF2 antibodies

  • Perform a dilution series to determine optimal signal-to-noise ratio for your specific sample type

• Blocking conditions:

  • 5% non-fat milk in TBST is typically sufficient

  • For phospho-specific detection, 5% BSA may yield better results

• Expected molecular weight:

  • CSTF2 appears at approximately 64 kDa

  • Verify this band is absent or reduced in CSTF2-knockout or knockdown controls

• Positive controls:

  • HeLa, Jurkat, HepG2, SW620 cell lysates are suitable positive controls

  • Mouse testis and thymus tissues also show strong CSTF2 expression

• Signal detection:

  • Both chemiluminescence and fluorescence-based detection systems are compatible

  • Longer exposure times may be needed for detecting endogenous levels in non-cancer cells

Remember that confirmation of antibody specificity through knockout or knockdown controls is critical for ensuring reliable results, especially when studying CSTF2 in cancer research applications.

How can I optimize CSTF2 antibody use for immunohistochemistry in cancer tissue samples?

Optimizing CSTF2 antibody performance in immunohistochemistry for cancer tissues requires careful attention to these methodological details:

• Tissue fixation and processing:

  • Optimal fixation in 10% neutral-buffered formalin for 24-48 hours

  • Paraffin embedding should follow standard protocols

  • Use freshly cut 4-5μm sections for best results

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) is typically effective

  • For difficult samples, test EDTA buffer (pH 9.0) as an alternative

  • Optimize retrieval time (15-30 minutes) based on tissue type

• Antibody incubation:

  • Begin with 1:100 dilution for most CSTF2 antibodies

  • Incubate overnight at 4°C for optimal sensitivity

  • Include controls: CSTF2-high tissues (HCC, lung cancer) and normal tissues

• Signal detection system:

  • Polymer-HRP systems typically provide superior signal-to-noise ratio

  • DAB is the recommended chromogen for CSTF2 detection

  • Counterstain with hematoxylin to visualize tissue architecture

• Validation approaches:

  • Peptide competition assays can confirm specificity

  • Use tissue with known CSTF2 status (e.g., confirmed by WB or qPCR)

  • Include CSTF2-knockout or knockdown tissues when available

• Interpretation guidelines:

  • CSTF2 shows primarily nuclear localization

  • In HCC, intensity correlates with histological grade and prognosis

  • Use digital imaging analysis for quantification when possible

When studying CSTF2 in cancer tissues, note that subcellular localization patterns may vary by cancer type and stage, making proper controls and validation critical for accurate interpretation.

How can I utilize CSTF2 antibodies to study its role in alternative polyadenylation and cancer progression?

To investigate CSTF2's role in alternative polyadenylation (APA) and cancer progression, implement these advanced methodological approaches:

• RNA immunoprecipitation (RIP) with CSTF2 antibodies:

  • Cross-link RNA-protein complexes using formaldehyde (1%) for 10 minutes

  • Immunoprecipitate with validated CSTF2 antibodies

  • Sequence precipitated RNAs to identify direct CSTF2 targets

  • Focus analysis on 3'UTR binding sites near polyadenylation signals

• Coupled CSTF2 ChIP-seq and 3'-seq analysis:

  • Perform chromatin immunoprecipitation with CSTF2 antibodies

  • In parallel, conduct 3'-seq to map polyadenylation sites

  • Integrate datasets to correlate CSTF2 binding with APA events

  • Compare patterns between normal and cancer cells to identify cancer-specific events

• CSTF2 knockdown/knockout coupled with 3'UTR reporter assays:

  • Design luciferase reporters containing target gene 3'UTRs with alternative polyadenylation sites

  • Measure reporter activity in CSTF2-depleted vs. control cells

  • Focus on cancer-relevant genes like HK2 in HCC studies

  • Include mutational analysis of CSTF2 binding sites

• Proximity ligation assays to study CSTF2 interactions:

  • Use CSTF2 antibodies alongside antibodies against other APA factors

  • Visualize and quantify protein-protein interactions in situ

  • Compare interaction patterns in normal vs. cancer tissues

  • Correlate with clinical parameters and patient outcomes

• Combined m6A-seq and CSTF2 RIP-seq:

  • Map m6A modifications genome-wide using m6A-specific antibodies

  • Correlate with CSTF2 binding patterns from RIP-seq

  • Focus analysis on cancer-relevant transcripts showing differential APA

  • This approach can reveal mechanisms like those found in PDAC, where CSTF2 regulates m6A installation

These integrated approaches enable comprehensive investigation of how CSTF2-mediated APA contributes to cancer progression, potentially revealing therapeutic vulnerabilities.

What techniques can I use to investigate CSTF2's role in hypoxia tolerance in hepatocellular carcinoma?

To investigate CSTF2's role in hypoxia tolerance in HCC, employ these methodological approaches:

• Hypoxia-specific CSTF2 protein analysis:

  • Culture HCC cells under normoxia (21% O₂) and hypoxia (1% O₂) conditions

  • Analyze CSTF2 protein levels by Western blot at multiple time points (4h, 8h, 24h)

  • Assess CSTF2 subcellular localization via immunofluorescence

  • Compare results across multiple HCC cell lines (e.g., Huh7, MHCC-97H, Hep3B)

• Metabolic profiling in CSTF2-modified HCC cells:

  • Measure extracellular acidification rate (ECAR) using Seahorse analyzer

  • Quantify glucose uptake and lactate production

  • Compare CSTF2 wild-type, knockout, and overexpressing cells under hypoxia

  • Focus on PGK1-dependent metabolic pathways

• 3'UTR analysis of glycolysis-related genes:

  • Perform 3'-seq to map polyadenylation sites in hypoxic vs. normoxic conditions

  • Focus on PGK1 and other glycolytic enzymes

  • Validate findings using qRT-PCR with primers specific to short vs. long 3'UTR isoforms

  • Correlate with m6A modification status

• In vivo hypoxia models:

  • Establish CSTF2-knockout and control HCC xenografts

  • Use pimonidazole staining to identify hypoxic regions

  • Analyze tumor growth rates and necrosis patterns

  • Correlate with glycolytic enzyme expression

• Integrated m6A modification analysis:

  • Perform m6A-seq under hypoxic conditions in CSTF2-modified cells

  • Analyze m6A reader (YTHDF2, YTHDC1) binding patterns

  • Focus on PGK1 and other metabolism-related transcripts

  • Validate findings using RNA stability assays

• Therapeutic targeting experiments:

  • Test masitinib as a CSTF2 inhibitor in hypoxic HCC models

  • Monitor effects on glycolytic activity and hypoxia tolerance

  • Evaluate combination with other metabolism-targeting agents

  • Assess efficacy in patient-derived organoid models

This comprehensive approach will elucidate how CSTF2 supports hypoxia tolerance in HCC through m6A modification and metabolic reprogramming, potentially revealing new therapeutic strategies.

How do I troubleshoot non-specific binding issues with CSTF2 antibodies?

When encountering non-specific binding with CSTF2 antibodies, implement this systematic troubleshooting approach:

• Verify antibody quality and storage:

  • Check antibody age and storage conditions (aliquoted at -20°C, minimal freeze-thaw cycles)

  • Run positive controls (HeLa, Jurkat, HepG2 lysates) alongside experimental samples

  • Consider testing a different antibody lot or manufacturer

• Optimize blocking conditions:

  • For Western blotting:

    • Test different blocking agents (5% milk, 5% BSA, commercial blockers)

    • Extend blocking time to 2 hours at room temperature

    • Add 0.1-0.3% Tween-20 to reduce background

  • For IHC/IF:

    • Include serum from the secondary antibody host species (2-5%)

    • Consider specialized blockers for endogenous biotin or peroxidase

    • Test commercial protein-free blockers

• Adjust antibody dilution and incubation:

  • Perform titration experiments (1:500 to 1:5000 for WB; 1:50 to 1:500 for IHC/IF)

  • Test longer incubation times at lower temperatures (overnight at 4°C)

  • Include 0.1-0.2% Tween-20 or 0.1% Triton X-100 in antibody diluent

• Improve washing protocols:

  • Increase wash duration (5-10 minutes per wash)

  • Add additional wash steps (minimum 3-5 washes)

  • Use agitation during washing

  • Include higher salt concentration (up to 500mM NaCl) to reduce ionic interactions

• Validate specificity with controls:

  • Include CSTF2 knockdown/knockout samples

  • Perform peptide competition assays with the immunizing peptide

  • Pre-adsorb antibody with cell/tissue lysates lacking CSTF2

• Application-specific considerations:

  • For IHC: Optimize antigen retrieval (citrate vs. EDTA buffers, pH, duration)

  • For IF: Test different fixation methods (4% PFA, methanol, acetone)

  • For WB: Adjust gel percentage and transfer conditions for 64kDa CSTF2

If non-specific binding persists despite these optimizations, consider using a monoclonal antibody, which may offer higher specificity though potentially lower sensitivity than polyclonal alternatives.

How can I interpret contradictory CSTF2 expression data between different experimental approaches?

When facing contradictory CSTF2 expression data between different experimental approaches, apply this systematic analytical framework:

• Antibody-related considerations:

  • Epitope differences: Different antibodies may target distinct CSTF2 regions, potentially detecting various isoforms or post-translationally modified variants

  • Application specificity: An antibody validated for Western blot may fail in IHC or IF applications

  • Batch variation: Compare lot numbers and validation data across experiments

• Protocol-specific variables:

  Method	Key Variables to Consider
  Western Blot	Sample preparation method, lysis buffer composition, protein denaturation conditions
  IHC	Fixation duration, antigen retrieval method, detection system sensitivity
  IF	Fixation method, permeabilization protocol, mounting medium autofluorescence
  qPCR	Primers targeting different transcript regions, reference gene stability

• Biological context analysis:

  • Cell type heterogeneity: CSTF2 expression varies between cell types; bulk tissue analysis may obscure cell-specific patterns

  • Experimental conditions: Hypoxia significantly affects CSTF2 function in HCC

  • Growth conditions: Cell confluence, serum starvation, and passage number can impact CSTF2 expression

• Transcript vs. protein discrepancies:

  • Investigate post-transcriptional regulation (miRNAs targeting CSTF2)

  • Assess protein stability and turnover rate in your experimental system

  • Consider analyzing both total CSTF2 and nuclear/cytoplasmic fractions separately

• Validation strategies:

  • Implement orthogonal techniques (e.g., mass spectrometry for protein, RNA-seq for transcript)

  • Use genetic approaches (siRNA, CRISPR) to validate antibody specificity

  • Perform cell-type specific analyses (laser capture microdissection, single-cell approaches)

• Data integration approach:

  • Weight evidence based on methodological rigor

  • Consider biological plausibility in light of known CSTF2 functions

  • Examine consistency with published literature on CSTF2 in similar contexts

When publishing such data, transparently report all methodological details and acknowledge limitations. This comprehensive analysis approach can transform apparently contradictory results into valuable insights about context-dependent CSTF2 regulation.

How can I effectively use CSTF2 antibodies in patient-derived cancer models?

For effective utilization of CSTF2 antibodies in patient-derived cancer models, implement these methodological approaches:

• Patient-derived xenograft (PDX) applications:

  • Perform dual immunostaining with human-specific CSTF2 antibodies and cancer markers

  • Validate antibody specificity using human vs. mouse-specific CSTF2 antibodies

  • Establish correlation between CSTF2 expression levels and PDX growth characteristics

  • Use serial sections to correlate CSTF2 with hypoxia markers (CA9, HIF1α) and glycolytic enzymes

• Patient-derived organoid (PDO) optimization:

  • Fix organoids using 4% PFA for 30 minutes followed by sucrose cryoprotection

  • Section at 8-10μm for optimal antibody penetration

  • Extend primary antibody incubation to 24-48 hours at 4°C

  • Use confocal microscopy with z-stack imaging for 3D expression analysis

  • Correlate with drug response profiles, particularly for masitinib

• Circulating tumor cell (CTC) analysis:

  • Optimize fixation protocols to preserve CSTF2 epitopes (2% PFA, 10 minutes)

  • Implement dual staining with epithelial markers (EpCAM, cytokeratins)

  • Consider microfluidic enrichment before immunostaining

  • Correlate CSTF2 expression with CTC viability and metastatic potential

• Ex vivo tissue slice culture applications:

  • Prepare 300μm precision-cut tissue slices from fresh tumor samples

  • Maintain in culture with defined media for up to 7 days

  • Apply CSTF2 antibodies in combination with viability indicators

  • Use for acute drug response studies with CSTF2 inhibitors

• Single-cell analysis integration:

  • Optimize tissue dissociation protocols to preserve epitopes

  • Implement multiparameter flow cytometry with CSTF2 antibodies

  • Correlate with stemness markers and metabolic indicators

  • Follow with single-cell RNA-seq to associate CSTF2 protein levels with transcriptomic profiles

• Clinicopathological correlation methodology:

  Parameter	Analysis Method	Expected Correlation
  Tumor grade	H-score quantification	Positive correlation in HCC
  Hypoxic regions	Pimonidazole co-staining	Enhanced CSTF2 in hypoxic zones
  Patient survival	Kaplan-Meier analysis	Negative correlation
  Treatment response	Pre/post-treatment comparison	Potential predictive biomarker

These approaches enable comprehensive characterization of CSTF2's role in patient-derived models, potentially identifying patient subsets who might benefit from CSTF2-targeted therapeutic strategies.

What are the current methodologies for evaluating CSTF2 as a therapeutic target in cancer?

Current methodologies for evaluating CSTF2 as a therapeutic target in cancer encompass these cutting-edge approaches:

• Genetic manipulation strategies:

  • CRISPR/Cas9-mediated CSTF2 knockout in cancer cell models

  • Inducible shRNA systems for temporal control of CSTF2 depletion

  • HTVI (hydrodynamic tail vein injection) for in vivo CSTF2 targeting in immunocompetent models

  • AAV-delivered CRISPR systems for organ-specific CSTF2 targeting

• Small molecule inhibitor screening:

  • High-throughput screening assays measuring CSTF2 RNA-binding capacity

  • AlphaScreen-based protein-protein interaction disruption assays

  • Structure-based virtual screening targeting CSTF2's RNA recognition motif

  • Repurposing screens identifying masitinib as a CSTF2 inhibitor

• Functional assessment methodologies:

  • Seahorse analysis for measuring glycolytic dependency post-CSTF2 inhibition

  • Colony formation assays under normoxic vs. hypoxic conditions

  • Invasion/migration assays correlating with CSTF2 expression levels

  • In vivo tumor formation assays with subcutaneous and orthotopic models

• Molecular mechanism evaluation:

  • 3'-seq for global APA profiling after CSTF2 modulation

  • m6A-seq to map methylation changes following CSTF2 inhibition

  • RNA Pol II elongation rate analysis using nascent RNA labeling techniques

  • RNA stability assays focusing on metabolism-related transcripts

• Preclinical efficacy assessment:

  • Patient-derived organoid drug response profiling

  • Combination studies with established therapies (sorafenib in HCC, gemcitabine in PDAC)

  • Immunocompetent models to assess effects on tumor microenvironment

  • Biomarker development for patient stratification

• Translational research approaches:

  Approach	Methodology	Expected Outcome
  Target engagement	Cellular thermal shift assay (CETSA)	Confirmation of direct CSTF2 binding by candidate molecules
  Pharmacodynamic markers	3'UTR length analysis of key targets	Reversal of CSTF2-mediated 3'UTR shortening
  Resistance mechanisms	Long-term inhibitor exposure	Identification of bypass pathways
  Patient selection	IHC-based scoring of CSTF2	Correlation with therapeutic response

These integrated methodologies provide a comprehensive framework for evaluating CSTF2 as a therapeutic target, potentially leading to novel cancer treatments targeting RNA processing vulnerabilities in multiple cancer types.