REXO2 Antibody

What is the molecular structure of REXO2 and what forms should be detected by antibodies?

REXO2 exists in multiple forms with distinct molecular weights that researchers should be aware of when interpreting western blot results. The full-length REXO2 precursor with mitochondrial targeting sequence has a predicted molecular weight of 26.8 kDa, while the mature mitochondrial form after cleavage of the targeting sequence is approximately 24 kDa. Interestingly, the cytosolic form has a similar mobility of about 24 kDa, potentially translated from an alternative start codon . When using Blue Native PAGE, researchers may detect higher molecular weight bands representing multimeric forms of REXO2 .

How can researchers validate the specificity of REXO2 antibodies?

For rigorous antibody validation:

• Compare protein detection in control vs. REXO2-depleted samples (using siRNA knockdown as described in the literature with sequences: sense 5′-GCAGGCAGAGUAUGAAUUUdTdT and antisense 5′-AAAUUCAUACUCUGCCUGCdTdT)

• Include purified recombinant REXO2 protein as a positive control

• Verify the expected molecular weight (approximately 24 kDa for mature REXO2)

• Perform subcellular fractionation to confirm dual localization to both cytosolic and mitochondrial compartments

• Treat isolated mitochondria with proteinase K to confirm that detected REXO2 is within mitochondria and not simply associated with the outer membrane

Which subcellular fraction markers should be used alongside REXO2 antibody?

When performing subcellular fractionation experiments with REXO2 antibody, include the following controls:

• Cytosolic markers: β-actin, S6

• Mitochondrial markers: porin (VDAC), AIF (inner mitochondrial membrane)

• Outer mitochondrial membrane: TOM20

• Mitochondrial matrix: GDH (glutamate dehydrogenase)

These markers help validate proper fractionation and confirm the dual localization of REXO2 to both cytosolic and mitochondrial compartments.

What is the optimal protocol for detecting REXO2 in western blotting?

Based on published methodologies:

• Sample preparation:

  • For whole cell lysates: Standard lysis buffers are suitable

  • For mitochondrial enrichment: Differential centrifugation followed by proteinase K treatment

• Gel electrophoresis:

  • For detecting individual forms: Standard SDS-PAGE

  • For capturing multimeric forms: Blue Native PAGE using 5%-18% gradient gels

• Transfer and detection:

  • Standard transfer protocols

  • Primary antibody: Custom-made anti-REXO2 or commercial antibodies

  • Secondary antibody: HRP-conjugated or fluorescent antibodies

  • Expected band: ~24 kDa for mature REXO2

How can researchers use REXO2 antibodies to study protein-protein interactions?

To investigate REXO2 interactions:

• Co-immunoprecipitation:

  • Lyse cells under gentle conditions to preserve protein complexes

  • Immunoprecipitate with anti-REXO2 antibody

  • Analyze precipitated complexes by western blotting

• Proximity labeling:

  • Generate REXO2 fusion proteins with BioID or APEX

  • Identify interaction partners by mass spectrometry

• Blue Native PAGE:

  • Use this technique to preserve native protein complexes

  • Western blot with anti-REXO2 antibody to identify multimeric forms and potential interaction partners

What protocols are recommended for studying REXO2 subcellular localization?

For accurate localization studies:

• Cell fractionation approach:

  • Isolate mitochondrial and cytosolic fractions through differential centrifugation

  • Analyze fractions by western blotting with anti-REXO2 antibody

  • Perform proteinase K treatment to confirm intramitochondrial localization

• Immunofluorescence microscopy:

  • Fix cells with paraformaldehyde

  • Stain with anti-REXO2 antibody and mitochondrial markers (e.g., TMRM+)

  • Analyze co-localization with confocal microscopy

How can REXO2 antibodies be used to investigate mitochondrial structure and function?

REXO2 antibodies can be applied to study multiple aspects of mitochondrial biology:

• Mitochondrial morphology:

  • Deplete REXO2 using siRNA

  • Monitor morphological changes using TMRM+ staining

  • Correlate with REXO2 protein levels using western blotting

• Mitochondrial membrane potential:

  • Perform time-lapse recordings of TMRM+ fluorescence

  • Analyze responses to inhibitors (oligomycin, rotenone, antimycin)

  • Correlate with REXO2 expression levels

• Mitochondrial nucleoid organization:

  • Visualize nucleoids using PicoGreen staining

  • Quantify nucleoid size and number in control vs. REXO2-depleted cells

  • Correlate with mtDNA copy number by qPCR

What role does REXO2 play in T cell function and how can antibodies help investigate this?

Recent findings highlight REXO2's importance in T cell anti-tumor activity:

• Expression analysis:

  • Compare REXO2 levels in different T cell subsets (particularly stem memory T cells)

  • Analyze REXO2 expression in T cells expanded with/without PI3Kδ inhibitor

• Metabolic studies:

  • Correlate REXO2 levels with metabolic fitness parameters

  • Measure spare respiratory capacity using Seahorse assays

  • Assess mitochondrial potential and ROS production

• Functional assessment:

  • Analyze how REXO2 knockout affects T cell anti-tumor potency in vivo

  • Investigate the relationship between REXO2 expression and T cell engraftment

How can researchers investigate REXO2's oligoribonuclease activity?

To study REXO2's enzymatic function:

• Activity assay setup:

  • Use fluorescently labeled (e.g., Alexa 647) oligoribonucleotides as substrates

  • Incubate with purified recombinant REXO2 or cell/mitochondrial lysates

  • Analyze reaction products using polyacrylamide gel electrophoresis

• Reaction conditions:

  • Buffer: 50 mM HEPES-KOH pH 7.4, 50 mM KCl, 10 mM MnCl2, 0.01% Triton X-100, 10% glycerol, 0.1 mM DTT

  • Temperature: 37°C

  • Reaction termination: Equal volume of 100% formamide, heated at 80°C for 3 minutes

• Product detection:

  • Separate products on 25% polyacrylamide gel without urea

  • Detect fluorescent products using phosphorimaging

  • Perform densitometric analysis

What are common challenges when using REXO2 antibodies and how can they be addressed?

Researchers often encounter these issues:

• Multiple band detection:

  • Challenge: Distinguishing between precursor (~26.8 kDa) and mature forms (~24 kDa)

  • Solution: Use subcellular fractionation to separate cytosolic and mitochondrial forms

• Low signal intensity:

  • Challenge: Insufficient antibody sensitivity

  • Solution: Optimize protein extraction, increase antibody concentration, or use enhanced chemiluminescence detection

• Non-specific binding:

  • Challenge: Background bands interfering with analysis

  • Solution: Increase blocking time/concentration, optimize antibody dilution, include REXO2-depleted samples as negative controls

How should researchers optimize detection of REXO2 in different experimental systems?

For optimal results across systems:

• Cell line variations:

  • Test antibody performance across multiple cell types

  • Adjust protein extraction methods based on cell type

  • Consider using inducible expression systems for low-expressing cells

• Tissue-specific considerations:

  • Modify extraction buffers to account for tissue composition

  • Increase clearing steps for lipid-rich tissues

  • Consider antigen retrieval methods for fixed tissues

• Animal model considerations:

  • Verify antibody cross-reactivity with the species of interest

  • Use Rexo2 knockout mice as negative controls

  • Adjust dilutions based on expression levels in different tissues

What controls are essential when investigating REXO2 function with antibodies?

Critical controls include:

• Positive controls:

  • Purified recombinant REXO2 protein

  • Cells overexpressing REXO2 (e.g., HEK293T cells with inducible REXO2-FLAG)

• Negative controls:

  • REXO2 siRNA-treated cells

  • Tissues from Rexo2 knockout mice

• Specificity controls:

  • Pre-absorption with recombinant protein

  • Secondary antibody-only controls

  • Isotype controls

How do alterations in REXO2 expression correlate with mitochondrial parameters?

REXO2 depletion studies reveal clear correlations:

• Mitochondrial morphology:

  • REXO2 depletion leads to punctate and granular mitochondria

  • This morphology correlates with mitochondrial dysfunction

• Mitochondrial membrane potential:

  • Control and REXO2-depleted cells show different responses to respiratory chain inhibitors

  • REXO2-depleted cells with stellate morphology become insensitive to rotenone

• Nucleic acid content:

  • REXO2 depletion causes mtDNA depletion (50-79% decrease)

  • Apparent disappearance of mitochondrial 7S DNA

  • Reduction in all mitochondrial RNA species with mt-mRNAs most affected

What impact does REXO2 depletion have on mitochondrial protein synthesis?

Depletion studies show progressive effects:

• Short-term effects (3 days):

  • Partial reduction in de novo synthesis of mtDNA-encoded proteins

  • Minimal change in steady-state levels of mitochondrial proteins

• Long-term effects (6 days):

  • Severely impaired mitochondrial protein synthesis

  • Appearance of aberrantly migrating polypeptides

  • Robust decrease in steady-state levels of mtDNA-encoded proteins (e.g., COX2) and nuclear-encoded respiratory complex subunits (e.g., NDUFB8)

How can researchers interpret REXO2 data in T cell-based cancer immunotherapy studies?

Recent findings provide these insights:

• T cell potency correlations:

  • REXO2 expression is elevated in stem memory T cells

  • REXO2 induction is linked to PI3Kδ signaling regulation

  • REXO2 knockout impairs T cell anti-tumor activity in vivo

• Metabolic fitness parameters:

  • REXO2 knockout leads to reduced spare respiratory capacity

  • Loss of REXO2 causes diminished mitochondrial potential

  • REXO2 deficiency results in increased reactive oxygen species production

• Clinical implications:

  • REXO2 status may predict treatment outcomes in adoptive T cell transfer therapy

  • REXO2 assessment could help determine the metabolic fitness of therapeutic T cells