PITHD1 Antibody

Basic Research Questions

• What is PITHD1 and why is it important in scientific research?

  PITHD1 (PITH domain-containing protein 1) is a 211 amino acid nuclear protein characterized by a single PITH domain that enables interaction with proteasome complexes. It functions as a proteasome-interacting protein essential for male fertility, acts as an endogenous inhibitor of the 26S proteasome, and plays a role in megakaryocyte differentiation.

  Research significance:

  • Essential for male reproductive function through proteasome regulation in testis

  • Functions as an endogenous proteasome inhibitor during cellular dormancy

  • Plays a critical role in megakaryocyte differentiation via RUNX1 regulation

  • Shows altered expression in leukemia and other cancer types

  • Induces pro-inflammatory phenotypes in specific cell types

  PITHD1 antibodies are essential tools for studying these diverse biological functions across different experimental systems and disease models .

• Which tissues and cell types express PITHD1, and how should expression analysis be approached?

  PITHD1 demonstrates highly tissue-specific expression patterns:

  Tissue/Cell Type	PITHD1 Expression	Detection Method
  Testis	High (specifically in spermatids)	RT-PCR, Immunoblot
  Thymus	High (specifically in cTECs)	RT-PCR, Immunoblot
  Olfactory neuroepithelial cells	Present	Mass spectrometry
  Normal peripheral blood MNCs	Relatively high	Immunoblot
  Leukemic cell lines (K562, HEL, etc.)	Low	Immunoblot
  Zebrafish oocytes and eggs	Present	Proteomic analysis

  Methodological approach for expression analysis:

  1. Quantitative RT-PCR to assess transcript levels (higher expression in testis than other organs)

  2. Immunoblotting with tissue fractionation to detect protein levels

  3. Immunohistochemistry with specific antibodies on tissue sections (using EDTA buffer pH 8.0 for antigen retrieval)

  4. Flow cytometry for cell-specific expression in mixed populations

  For optimal detection, use positive controls (testis or thymus tissue) and negative controls (PITHD1-deficient tissues) .

• What applications are PITHD1 antibodies validated for, and what are the optimal conditions?

  PITHD1 antibodies have been validated for multiple applications with specific optimal conditions:

  Application	Validation Status	Optimal Conditions
  Western Blotting (WB)	Validated	Fresh lysates with protease inhibitors
  Immunohistochemistry (IHC)	Validated	Heat-mediated antigen retrieval in EDTA buffer (pH 8.0); 2 μg/ml antibody concentration
  Immunofluorescence (IF)	Validated	Enzyme antigen retrieval; 5 μg/ml antibody concentration
  Immunoprecipitation (IP)	Validated	Effective for studying proteasome interactions
  ELISA	Validated	Test range: 0.156 ng/ml - 10 ng/ml

  For IHC, optimal results are achieved with overnight incubation at 4°C followed by appropriate secondary antibody detection systems. Most antibodies detect PITHD1 protein from human, mouse, and rat origins .

• How can I validate PITHD1 antibody specificity for my experiments?

  A methodical validation approach should include:

  1. Genetic controls:

     • Use tissues from PITHD1-knockout or PITHD1-deletion mice

     • Compare wild-type versus PITHD1-deficient samples by Western blot

     • Use siRNA or shRNA-mediated knockdown in cell lines

  2. Biochemical controls:

     • Conduct peptide competition assays with PITHD1 neutralizing peptide

     • Use multiple antibodies targeting different epitopes

     • Analyze molecular weight consistency (expected: ~25 kDa)

  3. Expression pattern validation:

     • Verify tissue-specific expression (high in testis and thymus)

     • Confirm subcellular localization patterns

     • Check for expected upregulation during differentiation (e.g., PMA-induced megakaryocyte differentiation)

  Research groups have successfully validated antibody specificity using CRISPR/Cas9-generated PITHD1-deficient mouse tissues (shown by PCR, Southern blot, and sequencing analyses) .

Advanced Research Questions

• How can I optimize protocols for studying PITHD1-proteasome interactions?

  For robust analysis of PITHD1-proteasome interactions:

  1. Co-immunoprecipitation protocol optimization:

     • Prepare fresh tissue lysates in buffer containing: 50 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100, protease inhibitors

     • Conduct IP with antibodies against specific proteasome subunits (α6, β5i, β5t, α4s)

     • Detect PITHD1 in precipitates by immunoblotting

     • Critical controls: IP with non-specific IgG, verification of proteasome subunit precipitation

  2. Tissue-specific considerations:

     • PITHD1 associates with β5i-containing immunoproteasomes in testis

     • PITHD1 does not associate with β5t-containing thymoproteasomes in thymus

     • PITHD1 does not associate with α4s-containing testis-specific proteasomes

  3. Functional assays:

     • Purified component assays using model substrates (e.g., sfGFP tagged with K48-linked polyubiquitin)

     • Measure proteasome activity with fluorogenic peptide substrates

     • Determine IC50 values for inhibition (approximately 302 ± 25 nM)

  4. Structural analysis:

     • Cryo-EM for visualizing PITHD1-proteasome complexes

     • Assess conformational changes in proteasome states (S1, S2, S3/4) .

• What are the key experimental approaches for investigating PITHD1's role in male fertility?

  A comprehensive investigation requires:

  1. Essential model systems:

     • PITHD1-deficient mice (knockout or deletion mutants)

     • Wild-type littermates as controls

     • Age-matched males for breeding experiments

  2. Fertility assessment protocol:

     • Breeding trials with wild-type females

     • Sperm count, morphology, and motility analyses

     • Hormonal profiling (testosterone, FSH, LH)

  3. Molecular and cellular analyses:

     Analysis	Method	Expected Findings in PITHD1-deficiency
     Testicular histology	H&E staining	Morphological abnormalities
     Sperm ultrastructure	Electron microscopy	Structural defects
     Proteasome activity	Fluorogenic substrate assay	Reduced activity in testis
     Fertilization proteins	Quantitative proteomics	Altered abundance of key proteins

  4. Validation approach:

     • Compare phenotypes between different PITHD1-deficient models (−/− and Δ/Δ)

     • Perform rescue experiments by reintroducing PITHD1

     • Examine proteasome composition and function in testis

  Research has demonstrated that PITHD1 deficiency leads to severe male infertility with morphological abnormalities and impaired motility of spermatozoa, while maintaining normal thymic function .

• How can I design experiments to study PITHD1's role in hematopoiesis and leukemia?

  To investigate PITHD1's function in megakaryocyte differentiation and leukemia:

  1. Experimental model systems:

     • K562 and HEL cell lines (leukemic cell models)

     • PMA treatment to induce megakaryocyte differentiation

     • Primary fetal liver cells for physiological relevance

     • Patient-derived leukemia samples

  2. Genetic manipulation approaches:

     • Lentiviral/retroviral transduction for PITHD1 overexpression

     • shRNA-mediated knockdown for loss-of-function studies

     • CRISPR/Cas9 for generating knockout cell lines

  3. Analytical methods:

     Analysis	Technique	Key Markers/Readouts
     Megakaryocyte differentiation	Flow cytometry	CD61, CD41 surface markers
     RUNX1 expression	Western blot, qRT-PCR	RUNX1 protein and mRNA levels
     Promoter activity	Dual luciferase assay	RUNX1 proximal promoter activity
     IRES activity	Dual luciferase assay	Internal ribosome entry site function

  4. Rescue experiment design:

     • Overexpress dominant negative RUNX1 (RUNX1 DN) to inhibit differentiation

     • Co-express PITHD1 to assess rescue capability

     • Measure CD61 expression and endogenous RUNX1 levels

  Research has shown that PITHD1 promotes megakaryocyte differentiation by enhancing RUNX1 expression through dual mechanisms: increasing transcription from the proximal promoter and enhancing translation via an IRES element in exon 3 .

• What are the optimal methods for investigating PITHD1 as a proteasome inhibitor?

  For characterizing PITHD1's inhibitory function:

  1. In vitro biochemical approaches:

     • Purified component assays with recombinant 26S proteasome

     • Model substrate degradation assays (e.g., polyubiquitinated GFP)

     • Dose-response studies with increasing PITHD1 concentrations

     • Analysis of DUB activity with isolated RPN8/RPN11 heterodimers

  2. Structure-function analysis:

     PITHD1 Variant	Structural Feature	Effect on Inhibition
     Full-length	Complete structure	Full inhibition (IC50 ~302 nM)
     PITHD1 1-198	Cannot enter central pore	Modest reduction in activity
     PITHD1 1-178	Cannot engage RPN11	Substantial decrease in inhibition

  3. Cellular approaches:

     • Generate cell lines with doxycycline-inducible PITHD1 expression

     • Monitor clearance of K48-ubiquitinated substrates

     • Compare with established proteasome inhibitors

     • Use E1 inhibitor (TAK-243) and DUB inhibitor (PR-619) for validation

  4. Conformational analysis:

     • Examine proteasome state distribution using cryo-EM

     • Analyze effects of PITHD1 on proteasome states (S1, S2, S3/4)

     • Study PITHD1's ability to drive proteasomes toward inactive conformations

  Research has revealed that PITHD1 functions through a "triple-lock" mechanism, simultaneously blocking three crucial functional sites on the 19S regulatory particle required for ubiquitin recognition, processing, and substrate translocation .

• How can I differentiate between PITHD1's interactions with different proteasome complexes?

  To distinguish PITHD1's selective binding to different proteasome types:

  1. Immunoprecipitation strategy:

     • Use antibodies against specific proteasome subunits:

       • Anti-β5t for thymoproteasomes

       • Anti-β5i for immunoproteasomes

       • Anti-α4s for testis-specific proteasomes

       • Anti-α6 for all proteasome complexes

     • Detect PITHD1 co-precipitation by immunoblotting

     • Include appropriate controls (IgG, input lysate)

  2. Critical experimental design elements:

     Proteasome Type	Tissue Source	Expected PITHD1 Binding	Control Verification
     Immunoproteasomes	Testis	Positive	Confirm β5i and α6 precipitation
     Thymoproteasomes	Thymus	Negative	Confirm β5t and α6 precipitation
     Testis-specific	Testis	Negative	Confirm α4s and α6 precipitation
     26S proteasome	Various	Positive	Confirm α6 precipitation

  3. Advanced visualization techniques:

     • Proximity ligation assay for in situ interaction detection

     • Fluorescence resonance energy transfer (FRET) for dynamic interactions

     • Cryo-EM for structural analysis of binding interfaces

  This methodical approach has revealed that PITHD1 selectively associates with β5i-containing immunoproteasomes in the testis but not with thymoproteasomes in the thymus or α4s-containing testis-specific proteasomes .

• What methodological approaches should be used to evaluate PITHD1 as a prognostic biomarker in cancer?

  For investigating PITHD1's potential as a cancer biomarker:

  1. Expression analysis methods:

     • Immunohistochemistry on tissue microarrays with appropriate controls

     • RT-qPCR for mRNA quantification across patient samples

     • Western blot for protein level assessment

     • Mining public gene expression databases (e.g., KM plotter)

  2. Statistical analytical framework:

     Statistical Method	Purpose	Example from Literature
     Univariable analysis	Initial assessment	HR = 0.22, P = 0.012 for OS
     Multivariable analysis	Adjusted for clinical parameters	HR = 0.082, P = 0.0013 for OS
     C-index calculation	Predictive performance measurement	Improved from 0.569 to 0.694
     Kaplan-Meier analysis	Survival curve comparison	Significant difference between high/low expression

  3. Validation requirements:

     • Independent patient cohorts with adequate sample sizes

     • Inclusion of established clinical parameters (age, stage, etc.)

     • Multivariate modeling to assess added predictive value

     • Consideration of tissue-specific expression patterns

  4. Functional correlation studies:

     • Assess relationship between expression and cellular phenotypes

     • Investigate mechanism linking PITHD1 to disease progression

     • Consider context-dependent effects (different in leukemia vs. solid tumors)

  Research has demonstrated that PITHD1 expression correlates with patient outcomes in ovarian cancer and is significantly downregulated in leukemia, suggesting potential utility as a prognostic biomarker in multiple cancer types .