PDCD2L Antibody

What is PDCD2L and what are its key functions in cellular biology?

PDCD2L (Programmed Cell Death Protein 2-like) is a shuttle protein that moves between the nucleus and cytoplasm. It has been identified with several key functions:

• Functions in ribosome biogenesis pathways

• Plays a regulatory role in inflammatory processes

• Involved in cell cycle regulation, particularly at S phase

• May contribute to programmed cell death mechanisms

• Regulates transcriptional activation of multiple pathways including AP1, CREB, NFAT, and NF-κB

The protein is known by several aliases including MGC13096 and is encoded by the PDCD2L gene (Entrez Gene ID: 84306). The human PDCD2L protein is identified by UniProt ID Q9BRP1 with a predicted band size of 39 kDa .

What experimental applications are PDCD2L antibodies validated for?

PDCD2L antibodies have been validated for multiple research applications:

• Western blot (WB): Typically used at dilutions of 1/1000 for detecting PDCD2L in human cell lysates including HeLa and MCF7 cell lines

• Immunohistochemistry on paraffin-embedded tissues (IHC-P): Successfully employed at 1/100 dilution for examining PDCD2L expression in human colon and liver cancer tissues

• Co-immunoprecipitation: Used for protein-protein interaction studies and mass spectrometry identification of binding partners

• Cellular thermal shift assays (CETSA): Applied for evaluating protein-small molecule interactions

When planning experiments, it's important to validate antibody specificity in your particular experimental system as performance may vary across different applications and species.

How does PDCD2L affect cell proliferation and cycle progression?

PDCD2L has demonstrated significant effects on cell proliferation and cell cycle progression:

• Overexpression of PDCD2L restrains proliferation of HEK293T cells

• DNA/flow cytometry analysis shows that PDCD2L overexpression severely delays cell cycle progression specifically at S phase

• The mechanisms may involve suppression of multiple transcription factors including AP1, CREB, NFAT, and NF-κB

For researchers studying cell cycle effects, flow cytometry with propidium iodide staining or BrdU incorporation assays are recommended methods to quantify cell cycle distribution changes following PDCD2L manipulation.

What is known about PDCD2L's role in inflammatory processes?

Recent research has established PDCD2L as a proinflammatory mediator:

• LPS stimulation tends to increase PDCD2L expression in cellular models

• Interference with PDCD2L expression reduces LPS-induced inflammation in vascular endothelial cells

• Key inflammatory markers affected include IL-6, IL-1β, and the adhesion factor ICAM1

• Multiple inflammatory mediators are reduced upon PDCD2L knockdown including CTACK, GRO-α, IFN-γ, IL-2Rα, IL-1α, IL-6, IL-8, MCP-3, MIP-1β, PDGF-BB, and TNF-α

• The regulatory mechanism appears to involve inflammatory transcription factors STAT1 and NF-κB

When designing experiments to investigate PDCD2L's inflammatory functions, consider using qPCR for mRNA expression analysis and Luminex or ELISA assays for cytokine protein detection in culture supernatants.

What methodological approaches are most effective for studying PDCD2L's role in ribosome biogenesis?

PDCD2L has been implicated in ribosome biogenesis, although detailed mechanisms require further investigation. Consider these methodological approaches:

• Sucrose gradient ultracentrifugation to isolate and analyze ribosomal subunits and their association with PDCD2L

• RNA-seq and ribosome profiling to assess changes in translation following PDCD2L manipulation

• Immunofluorescence co-localization studies with nucleolar markers

• Pulse-chase labeling with 5-fluorouridine to track rRNA synthesis

• Proximity ligation assays to detect PDCD2L interactions with ribosomal proteins or assembly factors

When investigating PDCD2L's function in ribosomes, consider siRNA-mediated knockdown approaches that have been successfully applied in EA.hy926 cells . Monitoring pre-rRNA processing intermediates by Northern blotting can provide insights into specific steps of ribosome biogenesis affected by PDCD2L.

How can researchers optimize co-immunoprecipitation protocols with PDCD2L antibodies?

For successful co-immunoprecipitation studies with PDCD2L antibodies, consider the following optimized protocol based on published methods:

• Prepare cell lysate in a buffer containing appropriate detergent (e.g., NP-40 or Triton X-100)

• Pre-clear lysate with agarose protein A+G beads to reduce non-specific binding

• Incubate PDCD2L antibody with agarose protein A+G at 4°C for 2 hours with gentle rotation

• Wash antibody-bound beads with PBS (3× recommended)

• Add pre-cleared cell lysate to antibody-bound beads and incubate overnight at 4°C

• Wash extensively with PBS (3× minimum)

• For mass spectrometry analysis:

  • Add DTT (10 mM) and incubate at a37°C for 2 hours

  • Add iodoacetamide (IAA, 50 mM) and incubate at room temperature in dark for 1 hour

  • Add trypsin for overnight digestion at 37°C

  • Desalt and dry samples before mass spectrometry analysis

This protocol has been successfully used to identify PDCD2L interaction partners. Key considerations include using appropriate controls such as IgG control antibodies and validating interactions with reciprocal co-IP or alternative methods.

What techniques are recommended for studying PDCD2L's interactions with small molecules?

Recent research has identified andrographolide (Andro) as a PDCD2L-binding molecule with anti-inflammatory effects. The following techniques have proven effective for studying PDCD2L-small molecule interactions:

• Differential Scanning Fluorimetry (DSF)

  • Mix purified PDCD2L protein with fluorescent probes

  • Measure thermal denaturation curves in the presence/absence of candidate compounds

  • A decrease in melting temperature (Tm) of 1.25°C was observed with 25 mM Andro

• Cellular Thermal Shift Assay (CETSA)

  • Analyze protein stability in cell lysates across temperature gradients

  • Monitor dose-dependent effects on protein stability

  • Both temperature and dose-dependent CETSA confirmed Andro's effect on PDCD2L thermal stability

• Competitive Binding Assays

  • Use IAA alkyne probes that bind to cysteine residues

  • Employ click chemistry to connect fluorescent reporters

  • Competition between small molecules and probes indicates binding site overlap

  • Andro showed competitive binding with IAA alkyne probes, suggesting cysteine-mediated interactions

• Molecular Docking

  • Utilize protein crystal structures (e.g., AF-Q9BRP1-F1-model_v4 from UniProt)

  • Software such as Autodock 4.2.6 for virtual docking

  • Visualize using PyMOL and Discovery Studio Client

  • Docking identified interactions via hydrogen bonding, van der Waals forces, and hydrophobic interactions, with cysteine 82 as a potential binding site

These complementary approaches provide robust validation of small molecule interactions with PDCD2L.

What experimental design is optimal for evaluating PDCD2L's effects on inflammatory pathways?

To thoroughly investigate PDCD2L's role in inflammation, consider this comprehensive experimental design:

• Cell Model Selection

  • Vascular endothelial cells (EA.hy926) have shown robust inflammatory responses to LPS with PDCD2L involvement

  • HEK293T cells are suitable for overexpression studies

  • Daudi cells demonstrate PDCD2L-mediated effects on TNF-alpha release

• PDCD2L Manipulation Approaches

  • siRNA knockdown: Use two different siRNAs targeting PDCD2L to control for off-target effects

  • Overexpression: Employ expression vectors with appropriate tags for detection

  • Include proper controls (scrambled siRNA, empty vector)

• Inflammatory Stimulation

  • LPS is well-validated as an inflammatory stimulus in this context

  • Consider time-course experiments (2, 4, 8, 24 hours)

  • Include positive control anti-inflammatory compounds (e.g., dexamethasone)

• Readout Methods

  • qPCR for gene expression (PDCD2L, ICAM1, IL-6, IL-1β)

  • Luminex multiplex assay for comprehensive cytokine/chemokine profiling

  • Western blot for protein expression and phosphorylation of STAT1 and NF-κB

  • ROS measurement for oxidative stress assessment

  • Analysis of antioxidant markers (CAT, GSH/GSSG, SOD)

• Mechanistic Investigation

  • Chromatin immunoprecipitation for transcription factor binding

  • RNA immunoprecipitation for mRNA transport analysis

  • Inhibitors of specific pathways to elucidate mechanism

Including all these elements provides a comprehensive assessment of PDCD2L's inflammatory functions and potential therapeutic targeting.

How should researchers assess PDCD2L's impact on oxidative stress markers?

PDCD2L has been shown to influence oxidative stress in vascular endothelial cells. For proper assessment of oxidative stress parameters in relation to PDCD2L function, consider these methodological approaches:

• ROS Production Measurement

  • Use fluorescent probes such as DCFDA (2',7'-dichlorofluorescein diacetate) for general ROS

  • MitoSOX Red for mitochondrial superoxide detection

  • Flow cytometry or fluorescence microscopy for quantification

  • Include positive controls such as H₂O₂ treatment

• Antioxidant System Analysis

  • Measure catalase (CAT) activity using colorimetric or fluorometric assays

  • Glutathione ratio (GSH/GSSG) using enzymatic recycling methods

  • Superoxide dismutase (SOD) activity assays

  • Quantify NRF2 pathway activation as a master regulator of antioxidant response

• Experimental Design Considerations

  • Compare PDCD2L knockdown versus overexpression

  • Include time-course analysis post-stimulation

  • Consider both basal and stimulated (e.g., LPS, TNF-α) conditions

  • Assess dose-dependent relationships

When overexpressed, PDCD2L has been shown to increase LPS-induced ROS production, reduce CAT and GSH/GSSG levels, and increase SOD levels . This pattern suggests a complex relationship with oxidative stress that may involve compensatory mechanisms.

PDCD2L Antibody Applications and Recommended Dilutions

Inflammatory Factors Regulated by PDCD2L

PDCD2L Interaction with Andrographolide (Andro)

How can I optimize detection of endogenous PDCD2L protein?

When detecting endogenous PDCD2L, researchers often encounter challenges due to expression levels or antibody sensitivity. Consider these optimization strategies:

• Use cell lines with documented PDCD2L expression (HeLa, MCF7)

• For Western blot, increase protein loading (50-100 μg) and optimize antibody concentration

• Enhance detection with high-sensitivity ECL substrates

• Enrich nuclear fraction for better detection, given PDCD2L's nuclear-cytoplasmic shuttling

• Consider immunoprecipitation followed by Western blot for low expression samples

• For IHC-P applications, optimize antigen retrieval methods (heat-induced epitope retrieval in citrate buffer has shown good results)

• Test multiple antibodies targeting different epitopes of PDCD2L

Proper negative controls (such as siRNA knockdown samples) are essential to confirm antibody specificity.

What controls are essential for PDCD2L functional studies?

Rigorous controls are crucial for meaningful interpretation of PDCD2L functional studies:

• For knockdown experiments:

  • Include non-targeting siRNA/shRNA controls

  • Use multiple siRNA sequences targeting different regions of PDCD2L

  • Validate knockdown efficiency at both mRNA (qPCR) and protein (Western blot) levels

  • Consider rescue experiments with siRNA-resistant PDCD2L expression

• For overexpression studies:

  • Include empty vector controls

  • Use expression tags that don't interfere with function

  • Verify expression by Western blot

  • Consider dose-response studies with varying expression levels

  • Include functional mutants (if known) as controls

• For inflammatory studies:

  • Include positive controls (e.g., dexamethasone for anti-inflammatory effects)

  • Perform time-course experiments for optimal response times

  • Include both unstimulated and stimulated conditions

• For protein interaction studies:

  • Use IgG control for co-immunoprecipitation

  • Include known interaction partners as positive controls

  • Validate with reciprocal IP experiments

  • Consider size exclusion chromatography as an orthogonal method

These controls ensure the specificity and reliability of your findings regarding PDCD2L functions.

What are promising research avenues for PDCD2L as a therapeutic target?

Given the emerging role of PDCD2L in inflammation and cell cycle regulation, several research directions show particular promise:

• Anti-inflammatory drug development

  • Andrographolide provides an initial scaffold for developing PDCD2L-targeting compounds

  • Structure-activity relationship studies could optimize binding and efficacy

  • High-throughput screening for additional PDCD2L-binding molecules

  • In vivo validation in inflammatory disease models

• Cancer therapeutic potential

  • Given PDCD2L's effect on cell cycle (S phase delay)

  • Investigate combinatorial approaches with existing chemotherapeutics

  • Explore context-dependent effects in different cancer types

  • Assess impact on cancer cell sensitivity to apoptosis

• Mechanistic investigations

  • Further elucidate the link between ribosome biogenesis and inflammation

  • Characterize the complete PDCD2L interactome under different conditions

  • Investigate post-translational modifications regulating PDCD2L function

  • Develop conditional knockout mouse models for in vivo studies

• Diagnostic applications

  • Evaluate PDCD2L as a biomarker in inflammatory diseases

  • Assess correlation between PDCD2L expression and disease progression

  • Investigate tissue-specific expression patterns in various pathologies

These research directions highlight the potential of PDCD2L as both a therapeutic target and a biological marker in inflammatory and proliferative disorders.