TP53INP2 Antibody, FITC conjugated

Basic Research Questions

• What is TP53INP2 and what cellular functions should researchers consider when designing experiments?

  TP53INP2 (Tumor protein p53-inducible nuclear protein 2, also known as DOR, C20orf110, PINH, or PIG-U) is a dual regulator of transcription and autophagy that functions as a scaffold protein in autophagosome formation .

  When designing experiments, researchers should consider:

  • TP53INP2's subcellular localization: It shuttles between nucleus and cytoplasm depending on cellular stress conditions and relocalizes to autophagosomes upon autophagy activation

  • Its key interactions with: MAP1LC3A, GABARAP, GABARAPL2, VMP1, and the BECN1-PI3-kinase class III complex

  • Its function as both an autophagic adaptor and transcriptional activator of THRA

  Methodological consideration: When investigating TP53INP2's dual functions, design experiments that can distinguish between its nuclear role in transcription and its cytoplasmic role in autophagy through appropriate subcellular fractionation or imaging approaches.

• What are the optimal applications for FITC-conjugated TP53INP2 antibodies?

  Based on reported applications for unconjugated TP53INP2 antibodies, FITC-conjugated variants would be most suitable for:

  • Immunofluorescence microscopy for tracking TP53INP2's translocation between nucleus and autophagosomes

  • Flow cytometry for quantitative analysis of TP53INP2 expression in heterogeneous cell populations

  • Immunocytochemistry for co-localization studies with autophagy markers like LC3

  Methodological table - Application-specific parameters:

  Application	Recommended Dilution	Incubation Time	Temperature	Key Controls
  Flow Cytometry	1:50-1:100	30 minutes	4°C	Isotype control (e.g., FITC-IgG)
  Immunofluorescence	1:100	1-2 hours	RT	Secondary-only, blocking peptide
  Co-localization Studies	1:50-1:100	1 hour	RT	Non-autophagy conditions

• How should I optimize staining protocols for FITC-conjugated TP53INP2 antibodies?

  For optimal results when using FITC-conjugated TP53INP2 antibodies:

  1. Sample fixation: Use 4% paraformaldehyde for 15 minutes at room temperature

  2. Permeabilization: 0.1% Triton X-100 for 10 minutes is suitable for accessing both nuclear and cytoplasmic TP53INP2

  3. Blocking: Use 5% normal serum from the same species as the secondary antibody for at least 30 minutes

  4. Antibody dilution: Start with manufacturer's recommendation, typically 1:50-1:100 dilution

  5. Incubation: Overnight at 4°C or 1-2 hours at room temperature

  6. Photobleaching prevention: Mount with anti-fade reagent containing DAPI for nuclear counterstaining

  Critical optimization step: Since TP53INP2 shuttles between nucleus and cytoplasm based on cellular conditions, the fixation method can significantly affect observed localization patterns. Compare multiple fixation protocols if discrepancies are observed .

• What validation approaches should be employed when using TP53INP2 antibodies in new experimental systems?

  Before relying on results from FITC-conjugated TP53INP2 antibodies:

  1. Perform knockdown/knockout validation: Use siRNA against TP53INP2 (demonstrated effective in references ) to verify signal specificity

  2. Peptide competition assay: Pre-incubate antibody with immunizing peptide to confirm binding specificity

  3. Cross-validate with independent antibody clones targeting different epitopes

  4. Confirm expected molecular weight (~23kDa) by Western blot if using unconjugated version of the same antibody clone

  5. Verify expected subcellular localization patterns under different conditions (nuclear in basal conditions, cytoplasmic/autophagosomal during autophagy induction)

  Methodological insight: When validating TP53INP2 antibodies for autophagy studies, include both autophagy-inducing conditions (starvation, rapamycin treatment) and inhibition conditions (chloroquine, wortmannin) to confirm expected localization changes .

Advanced Research Questions

• How can I design experiments to differentiate between TP53INP2's role in autophagy versus apoptosis pathways?

  TP53INP2 has been shown to function in both autophagy and apoptosis pathways, making experimental design crucial:

  1. Sequential inhibition approach:

     • Block autophagy using ATG5/ATG7 knockdown or pharmacological inhibitors like wortmannin

     • Analyze apoptotic response through TP53INP2-dependent caspase-8/TRAF6 pathways

  2. Domain-specific mutations:

     • Use TP53INP2 W35,I38A LIR double mutant (incapable of binding LC3) to specifically disrupt autophagy function

     • Compare with wild-type TP53INP2 to delineate autophagy-dependent versus autophagy-independent functions

  3. Pathway-specific readouts:

     • Autophagy: LC3-II/LC3-I ratio, p62 degradation, autophagosome formation by TEM

     • Apoptosis: Caspase-3/8 activation, PARP cleavage, annexin V staining

  Key experimental insight: TP53INP2 induces apoptosis in clear cell renal cell carcinoma through the caspase-8/TRAF6 pathway, independent of its autophagy function . Use chloroquine treatment alongside TP53INP2 manipulation to distinguish between these pathways.

• What approaches reveal the interaction dynamics between TP53INP2 and autophagy-related proteins?

  To study interaction dynamics between TP53INP2 and autophagy machinery:

  1. Co-immunoprecipitation with staged autophagy induction:

     • Perform time-course experiments following starvation or rapamycin treatment

     • IP with anti-TP53INP2 antibody and probe for ATG7, LC3, GABARAP, GABARAPL2

  2. Bioluminescence resonance energy transfer (BRET) assays:

     • BRET has demonstrated that interactions between TP53INP2 and LC3/GABARAP proteins require autophagy and are modulated by wortmannin

     • Design BRET constructs with TP53INP2 and potential binding partners

  3. In vitro affinity-isolation assays:

     • Use purified recombinant GST-TP53INP2 or GST-TP53INP2 W35,I38A

     • Assess direct binding to purified LC3B[G120] and ATG7

  4. Proximity ligation assay (PLA) for endogenous protein interactions:

     • Use antibody pairs targeting TP53INP2 and binding partners

     • Visualize interaction dynamics during autophagy progression

  Advanced research consideration: Studies show that TP53INP2 interacts directly with ATG7 even when its LC3-binding ability is disrupted through W35,I38A mutations, suggesting multiple interaction interfaces with the autophagy machinery .

• How can I investigate TP53INP2's function as an autophagic adaptor for ubiquitinated proteins?

  To study TP53INP2's role in ubiquitinated protein degradation:

  1. Identification of the ubiquitin-interacting motif (UIM):

     • Generate TP53INP2 constructs lacking the UIM

     • Assess binding to ubiquitin and ubiquitinated proteins via pull-down assays

  2. Competitive displacement assays:

     • Determine if TP53INP2 lacking UIM displaces p62 from LC3

     • Monitor accumulation of ubiquitinated proteins

  3. Sensitivity to autophagy inhibition:

     • Compare cells expressing WT versus UIM-deleted TP53INP2

     • Assess sensitivity to chloroquine treatment

  4. Autophagic substrate degradation assays:

     • Track degradation of known ubiquitinated autophagy substrates

     • Compare degradation kinetics in presence of WT versus mutant TP53INP2

  Research finding: TP53INP2 lacking the UIM can displace autophagic adaptor p62 from LC3, leading to accumulation of ubiquitinated proteins in cells, and sensitizes cells to chloroquine treatment .

• What methodological approaches can evaluate TP53INP2 as a prognostic biomarker in cancer research?

  For investigating TP53INP2 as a cancer biomarker:

  1. Tissue microarray analysis:

     • Optimize IHC protocols for TP53INP2 detection in paraffin-embedded tissues

     • Correlate expression with clinical outcomes and pathological parameters

  2. Survival analysis approaches:

     • Stratify patients into TP53INP2-high and TP53INP2-low groups

     • Use Kaplan-Meier analysis and Cox proportional hazards models

  3. Multi-omics integration:

     • Correlate TP53INP2 expression with:

       • RNA-seq transcriptome profiles

       • miRNA expression patterns

       • Copy number variations

       • Mutation profiles

  4. Experimental validation in cancer models:

     • Assess impact of TP53INP2 manipulation on:

       • Cell proliferation using CCK-8 assays

       • Migration via wound scrape assays

       • Invasion through transwell assays

       • Apoptosis by flow cytometry

  Research insight: In head and neck squamous cell carcinoma, lower TP53INP2 expression correlates with poor prognosis. Patients with higher TP53INP2 expression showed longer survival time, and knockdown of TP53INP2 promoted cell viability .

• How can I develop multi-color flow cytometry panels incorporating FITC-conjugated TP53INP2 antibodies?

  For designing multi-parameter flow cytometry panels:

  1. Panel design considerations:

     • FITC emission spectrum (peak ~520nm) - avoid PE (575nm) in adjacent channels

     • Compatible fluorochromes: APC (660nm), BV421 (421nm), PerCP (675nm)

  2. Sample processing protocol:

     • Fix cells in 4% paraformaldehyde for 15 minutes

     • Permeabilize with 0.1% saponin or 0.1% Triton X-100

     • Block with 5% normal serum for 30 minutes

     • Incubate with antibodies for 30 minutes at 4°C

  3. Autophagy-focused panel example:

     Target	Fluorochrome	Purpose	Concentration
     TP53INP2	FITC	Primary protein of interest	1:50
     LC3	APC	Autophagosome marker	1:100
     p62	BV421	Autophagy substrate	1:100
     Cleaved Caspase-3	PerCP	Distinguish apoptosis	1:50

  4. Controls and compensation:

     • Single-stained controls for each fluorochrome

     • FMO (Fluorescence Minus One) controls

     • Isotype controls matched to antibody concentration

     • Autophagy-positive control (starved/rapamycin-treated cells)

     • Autophagy-negative control (wortmannin-treated cells)

  Methodological note: When analyzing autophagy by flow cytometry, including chloroquine-treated samples helps distinguish between increased autophagosome formation and impaired autophagosome clearance .

• What experimental designs can assess TP53INP2's role in muscle autophagy and sarcopenia?

  To investigate TP53INP2 in muscle biology and aging:

  1. Human muscle biopsy analysis:

     • Compare TP53INP2 protein levels between young and aged subjects

     • Correlate with hand-grip strength and physical performance metrics

     • Analyze relationship with Charlson comorbidity index (CCI)

  2. Mouse model experimental design:

     • Compare young (4-6 months) and old (22-24 months) transgenic mice

     • Measure autophagic flux using colchicine (0.4 mg/kg for two days)

     • Assess mitophagy by measuring LC3-II in mitochondrial fractions

  3. Molecular readouts to assess:

     • Autophagosome quantification in muscle sections

     • LC3-II/LC3-I ratio analysis

     • Expression of autophagy genes

     • Mitochondrial quality and function parameters

  4. Functional assessments:

     • Muscle strength measurements

     • Physical performance tests

     • Metabolic parameters

  Research finding: High TP53INP2 protein levels are associated with greater muscle strength, physical performance, and healthy aging in humans. In mouse models, TP53INP2 overexpression increases mitophagy, reduces mitochondrial mass and ROS production, maintaining mitochondrial respiration and improving metabolic homeostasis in aged animals .

• How can I optimize co-localization studies between TP53INP2 and other autophagy proteins?

  For high-quality co-localization experiments:

  1. Sample preparation considerations:

     • Timing: Co-localization is dynamic; create a time course after autophagy induction

     • Fixation: 4% paraformaldehyde preserves autophagosome structures

     • Permeabilization: Gentle (0.1% Triton X-100) to maintain organelle integrity

  2. Microscopy setups:

     • Confocal microscopy with appropriate channel separation

     • Super-resolution techniques (STED, SIM) for detailed co-localization

     • Live-cell imaging to track dynamic interactions

  3. Key protein pairs to examine:

     • TP53INP2 with LC3/GABARAP family proteins

     • TP53INP2 with VMP1 (at autophagosome membrane)

     • TP53INP2 with ATG7 (direct interaction demonstrated)

     • TP53INP2 with TRAF6 and caspase-8 (for apoptosis studies)

  4. Quantitative co-localization analysis:

     • Pearson's correlation coefficient

     • Manders' overlap coefficient

     • Object-based co-localization for punctate structures

  Experimental insight: Studies show that RFP-TP53INP2 has a diffuse cytoplasmic distribution in ATG5-/- and ATG7-/- MEFs and fails to co-localize with GFP-ATG14 puncta, indicating that autophagy machinery is required for TP53INP2's localization pattern .

• What strategies can investigate TP53INP2's therapeutic relevance in TRAIL-based cancer treatments?

  To study TP53INP2's role in TRAIL sensitivity:

  1. Patient stratification approach:

     • Analyze TP53INP2 expression in tumor samples

     • Correlate with response to TRAIL therapy

     • Identify threshold expression levels for response prediction

  2. Mechanistic investigations:

     • Assess TP53INP2's interaction with caspase-8 and TRAF6

     • Measure TRAF6-induced ubiquitination and activation of caspase-8

     • Evaluate impact on TRAIL-induced apoptosis pathways

  3. Therapeutic combination testing:

     • TRAIL monotherapy versus TRAIL combined with autophagy inhibitors

     • Compare effects in TP53INP2-high versus TP53INP2-low cells

     • Measure apoptotic markers and cell death kinetics

  4. In vivo validation studies:

     • Cell line-derived xenografts with varying TP53INP2 levels

     • Patient-derived xenografts stratified by TP53INP2 expression

     • Treatment with TRAIL alone or in combination therapies

  Clinical research finding: TP53INP2 significantly enhances TRAIL-induced apoptosis, especially in AML cells with nucleophosmin 1 (NPM1) mutations. Cytoplasmic TP53INP2 maintained by mutant NPM1 acts as a scaffold bridging TRAF6 to caspase-8, promoting ubiquitination and activation of the caspase-8 pathway .