PIH1D3 Antibody

What is PIH1D3 and why is it important for ciliary research?

PIH1D3 (PIH1 domain-containing protein 3) is a cytoplasmic protein involved in the preassembly of axonemal dynein arms, which are essential for motile cilia and flagella function. Mutations in PIH1D3 cause X-linked primary ciliary dyskinesia (PCD), characterized by chronic airway disease, infertility, and left-right body axis disturbance . PIH1D3 specifically contributes to the assembly of both outer dynein arms (ODAs) and inner dynein arms (IDAs), with loss-of-function mutations leading to their absence or reduction and consequently causing ciliary and flagellar immotility . The protein's importance has been established through studies demonstrating its interaction with cytoplasmic ODA/IDA assembly factors DNAAF2 and DNAAF4, as well as with chaperone proteins HSP70 and HSP90 .

Which tissue types express PIH1D3 and where should I expect positive antibody staining?

PIH1D3 expression is primarily observed in tissues containing motile ciliated cells. Immunohistochemical studies have demonstrated positive staining in:

PIH1D3 protein is specifically localized within the cytoplasm of these cells, with strong staining observed in proximity to the nucleus, but does not co-localize with ciliary basal body or axonemal markers . In spermatogenic cells, PIH1D3 is most abundant in pachytene spermatocytes but is not detected in spermatids or mature sperm .

What are the recommended fixation methods for optimal PIH1D3 immunostaining?

For optimal immunohistochemical detection of PIH1D3, the following fixation and antigen retrieval protocols are recommended:

• Fix tissue samples in 4% paraformaldehyde for 24 hours at room temperature.

• For paraffin-embedded sections, perform antigen retrieval using either:

  • TE buffer (pH 9.0) as the primary recommended method

  • Alternatively, citrate buffer (pH 6.0) can be used

• For immunofluorescence of cultured cells, fixation with 4% paraformaldehyde for 10 minutes at room temperature is suitable.

The antibody performs best when tissue sections are processed within 3 months of sectioning to prevent antigen degradation. For respiratory epithelial cells, sequential monolayer-suspension cell culture may be necessary before antibody application to maintain ciliary structures .

How can I distinguish between true PIH1D3 antibody staining and potential cross-reactivity in my model organism?

This question requires careful consideration due to the complex evolutionary history of PIH1D3. In humans, PIH1D3 is encoded by a single X-linked gene, while mice have two homologous copies located on chromosomes 1 and X . When working with model organisms, consider these approaches:

• RNA interference validation: Use siRNA knockdown of PIH1D3 in human cells or CRISPR-Cas9 genome editing to create controls for antibody specificity testing.

• Species-specific considerations:

  • Human samples: Most commercial PIH1D3 antibodies target human PIH1D3 (UNIPROT ID: Q9NQM4) .

  • Mouse models: Consider that mice have two PIH1D3 homologs with 91% protein identity but differences in the unstructured N-terminus . The X-linked mouse gene contains introns, while the chromosome 1 gene (often termed Pih1d3, 4930521A18Rik) is intronless and exclusively expressed in testis .

  • Rat models: Unlike mice, rats have only one copy of PIH1D3 located on the X chromosome, making them potentially better models for human PIH1D3-related pathologies .

• Control experiments: Always include:

  • Positive controls from tissues known to express PIH1D3 (lung, testis)

  • Negative controls using isotype-matched antibodies

  • PIH1D3-knockout tissues when available (though note that complete knockouts may be embryonic lethal in some models)

• Western blot validation: Confirm the specificity of your antibody by western blot, verifying that it detects a protein of the expected molecular weight (approximately 24 kDa) .

What are the methodological differences when using PIH1D3 antibodies for patients with PIH1D3 mutations versus controls?

When studying patient samples with PIH1D3 mutations, several methodological adjustments are necessary:

• Selection of appropriate antibody epitopes: Different PIH1D3 mutations affect different regions of the protein. For example:

  • Nonsense mutations (e.g., p.Glu43*, p.Trp89*, p.Gln171*) may result in truncated proteins or nonsense-mediated decay

  • Frameshift mutations (e.g., p.Ile88Argfs12, p.Ile164Leufs11) produce altered C-terminal sequences

  • Genomic deletions may eliminate the PIH1D3 gene entirely

  Therefore, select antibodies targeting protein regions upstream of common mutations or consider using multiple antibodies targeting different regions.

• Optimization of antibody dilutions: For patients with reduced PIH1D3 expression, you may need to adjust dilutions:

  • For controls: Use standard dilutions (1:50-1:500 for IHC)

  • For patient samples: Consider using more concentrated antibody solutions (1:20-1:100)

• Signal amplification techniques: For detecting residual PIH1D3 in patient samples:

  • Tyramide signal amplification may enhance sensitivity

  • Longer exposure times for immunofluorescence imaging

  • Use of high-sensitivity detection systems

• Control selection: Include both:

  • Healthy controls (normal PIH1D3 expression)

  • Disease controls (PCD patients with mutations in other genes) to distinguish PIH1D3-specific effects

• Complementary techniques: Confirm antibody results with:

  • mRNA expression analysis (RT-PCR or RNA-Seq)

  • Protein quantification by western blot

  • Transmission electron microscopy (TEM) of cilia to assess dynein arm defects

How can I optimize co-immunoprecipitation protocols to study PIH1D3 protein-protein interactions?

PIH1D3 interacts with several proteins involved in ciliary dynein assembly. To optimize co-immunoprecipitation (co-IP) protocols:

• Lysis buffer optimization:

  • Use a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, supplemented with complete protease inhibitor cocktail

  • For detecting weaker interactions, consider milder detergents (0.5% NP-40 or 0.1% Triton X-100)

  • Include phosphatase inhibitors if studying phosphorylation-dependent interactions

• Tag selection for recombinant proteins:

  • C-terminal FLAG-tagged PIH1D3 has been successfully used in previous studies

  • C-terminal HA-tagged potential interacting partners (DNAAF2, DNAAF4) also work well

  • Avoid N-terminal tags which may interfere with protein-protein interactions

• Validated interaction partners to use as positive controls:

  Protein	Interaction Strength	Detection Method
  DNAAF2/KTU	Strong	Co-IP and Y2H
  DNAAF4/DYX1C1	Strong	Co-IP and Y2H
  HSP90	Moderate	Co-IP
  HSP70	Moderate	Co-IP

• Optimization for specific interactions:

  • For HSP70/HSP90 interactions: Include 5 mM ATP in lysis buffer

  • For DNAAF2/DNAAF4: Include 1 mM DTT to maintain reducing conditions

  • For detecting complexes with dynein components: Longer incubation times (3-4 hours) at 4°C may be necessary

• Validation approaches:

  • Reciprocal co-IPs (use each protein as bait)

  • Confirmation with endogenous proteins (not just overexpressed)

  • Mass spectrometry to identify novel interactors

  • Proximity ligation assay for in situ validation

How can PIH1D3 antibodies be used to characterize primary ciliary dyskinesia patient samples?

PIH1D3 antibodies are valuable tools for diagnosing and characterizing X-linked PCD caused by PIH1D3 mutations:

• Diagnostic application workflow:

  • Obtain nasal or bronchial epithelial cells from patients with suspected PCD

  • Perform immunofluorescence staining with PIH1D3 antibody (cytoplasmic localization)

  • In parallel, stain for dynein arm markers DNAH5 (ODA marker) and DNALI1 (IDA marker)

  • Compare to control samples to assess PIH1D3 expression and dynein arm status

• Interpretation of staining patterns:

  Pattern	Interpretation	Follow-up
  Reduced/absent PIH1D3 + normal dynein markers	Possible partial PIH1D3 function	Genetic testing
  Reduced/absent PIH1D3 + reduced/absent DNAH5/DNALI1	Consistent with PIH1D3-related PCD	Genetic testing
  Normal PIH1D3 + reduced/absent dynein markers	PCD likely caused by other genes	Screen other PCD genes

• Complementary analyses:

  • Transmission electron microscopy to confirm ODA/IDA defects

  • High-speed video microscopy to assess ciliary beating

  • Immunoblotting for PIH1D3 and dynein components

  • Genetic analysis targeting PIH1D3 and other PCD genes

• Research applications:

  • Characterize novel PIH1D3 variants to determine pathogenicity

  • Correlate PIH1D3 expression levels with severity of ciliary ultrastructural defects

  • Compare cellular phenotypes between different PIH1D3 mutations

Case study: In a series of PIH1D3-mutation patients, PIH1D3 antibody staining showed varying degrees of protein reduction, correlating with the severity of dynein arm defects. Patient GVA30 II:1 (p.Ile164Leufs11) showed undetectable PIH1D3 levels, while patient PCD12 II:1 (p.Glu43) showed very faint staining, suggesting residual protein expression .

What methodological approaches can resolve contradictory data between PIH1D3 localization in human versus mouse models?

Resolving species-specific differences in PIH1D3 localization requires careful experimental design:

• Understanding species differences:

  • Humans have one X-linked PIH1D3 gene

  • Mice have two homologs: an intron-containing X-linked gene and an intronless chromosome 1 pseudogene

  • Rats have only one X-linked PIH1D3 gene, similar to humans

• Resolving contradictory localization data:

  • Use antibodies targeting conserved epitopes between species

  • Perform paralog-specific knockdown in mice to determine contribution of each gene

  • Generate species-specific antibodies when necessary

  • Include appropriate controls for each species

• Experimental design for comparative studies:

  • Use identical tissue processing methods across species

  • Apply the same antibody concentrations and incubation conditions

  • Image samples using identical microscope settings

  • Quantify fluorescence intensity using standardized methods

• Cell-type specific analysis:

  • In mouse testis: PIH1D3 is expressed in spermatogenic cells but not in spermatids or mature sperm

  • In human respiratory cells: PIH1D3 shows strong cytoplasmic localization near the nucleus

  • Compare similar cell types and developmental stages between species

• Validation approaches:

  • Confirm antibody specificity using knockout tissues from each species

  • Use epitope-tagged PIH1D3 expression in cell lines

  • Employ multiple antibodies targeting different PIH1D3 regions

  • Complement with mRNA localization studies (in situ hybridization)

Key research finding: Knockout mice lacking the testis-expressed chromosome 1 pseudogene (Pih1d3) show immotile sperm flagella with dynein arm defects but do not manifest other ciliary phenotypes typical of PCD such as situs inversus, respiratory cilia dysfunction, or hydrocephalus . This contrasts with human PIH1D3 mutations, which cause full PCD syndrome, suggesting functional differences between species .

What experimental design best evaluates the specificity of PIH1D3 antibodies across multiple applications?

A comprehensive validation approach should include:

• Multi-technique validation matrix:

  Technique	Positive Control	Negative Control	Expected Result
  Western Blot	Human bronchial epithelial lysate	PIH1D3 knockout/knockdown cells	Single band at 24 kDa
  IHC	Human lung tissue	Non-ciliated tissue (e.g., liver)	Cytoplasmic staining in ciliated cells
  IF	Human nasal epithelial cells	PIH1D3 siRNA-treated cells	Cytoplasmic punctate pattern
  IP	Overexpressed FLAG-tagged PIH1D3	Empty vector transfection	Enrichment of PIH1D3 and interactors

• Peptide competition assay:

  • Pre-incubate PIH1D3 antibody with excess immunizing peptide

  • Apply to duplicate samples alongside non-blocked antibody

  • Specific staining should be eliminated by peptide blocking

• Orthogonal validation using RNA analysis:

  • Compare antibody signal with mRNA expression data

  • Use RNA-seq or qPCR to confirm expression patterns

  • This approach is supported by enhanced validation data for some commercial antibodies

• Cross-application consistency analysis:

  • Test the same antibody across multiple applications

  • Compare localization patterns between IF and IHC

  • Verify protein size consistency between IP and WB

• Genetic manipulation controls:

  • Use CRISPR-Cas9 PIH1D3 knockout cells/tissues

  • Compare with siRNA knockdown samples

  • Include rescue experiments with wild-type PIH1D3

Example validation data: Some commercially available PIH1D3 antibodies have undergone enhanced validation using orthogonal RNAseq approaches , confirming specificity of detection. For immunohistochemistry applications, these antibodies show positive staining in human lung and testis tissues, with negative or minimal staining in non-ciliated tissues .

What controls should be included when studying PIH1D3 in ciliopathy models using knock-out animal systems?

When studying PIH1D3 in animal models of ciliopathy, include these essential controls:

• Genotype verification controls:

  • PCR confirmation of knockout status

  • RT-PCR to verify absence of PIH1D3 mRNA

  • Western blot to confirm absence of PIH1D3 protein

  • For conditional knockouts, verify tissue-specific deletion

• Animal model selection considerations:

  • Rat models may better reflect human PIH1D3 function than mouse models

  • PIH1D3-knockout rats reproduce cardinal features of ciliopathy including situs inversus, defects in spermatocyte survival, mucociliary clearance, and perinatal hydrocephalus

  • Mouse Pih1d3 knockouts show more limited phenotypes due to gene redundancy

• Phenotypic controls:

  • Wild-type littermates as primary controls

  • Heterozygous animals (especially for X-linked genes)

  • Age and sex-matched controls

  • For X-linked PIH1D3, include both male and female animals for analysis

• Functional assays with corresponding controls:

  Assay	Purpose	Controls
  Ciliary beat frequency	Assess motility	Compare to non-ciliated cells
  Electron microscopy	Evaluate dynein arms	Use standardized sections
  Mucociliary clearance	Assess in vivo function	Include known PCD models
  Immunofluorescence	Localize dynein components	Include multiple dynein markers

• Rescue experiments:

  • Reintroduce wild-type PIH1D3 cDNA

  • Use tissue-specific promoters

  • Create point mutation variants to test specific functions

  • Quantify degree of phenotypic rescue

Research finding: In PIH1D3-knockout rats, electron microscopy revealed that motile cilia lacked outer dynein arms and showed reduced inner dynein arms, reproducing the ultrastructural defects observed in human PCD patients with PIH1D3 mutations . This validates the rat as an appropriate model for human PIH1D3-related ciliopathies.

How should researchers interpret quantitative differences in PIH1D3 antibody staining between experimental conditions?

Proper interpretation of quantitative differences in PIH1D3 staining requires systematic analysis:

• Standardized quantification methods:

  • Use identical imaging parameters across all samples

  • Apply automated analysis algorithms to eliminate bias

  • Normalize PIH1D3 signals to housekeeping proteins or total protein

  • Include internal standards for cross-experiment comparison

• Statistical approach to quantification:

  • Analyze multiple fields per sample (minimum 5-10)

  • Include biological replicates (n≥3)

  • Apply appropriate statistical tests based on data distribution

  • Consider using ANOVA for multiple condition comparisons

• Interpretation of staining patterns:

  • Reduced intensity: May indicate partial loss of function or protein destabilization

  • Altered localization: Could suggest defective protein trafficking or complex formation

  • Complete absence: Likely indicates null mutation or protein degradation

  • Variable expression: May reflect mosaic expression or technical variability

• Correlation with functional outcomes:

  • Associate PIH1D3 levels with ciliary beat frequency

  • Correlate with dynein arm assembly status

  • Link to clinical severity in patient samples

  • Compare with other dynein assembly factors

• Technical considerations affecting quantitation:

  • Antibody lot-to-lot variation

  • Tissue fixation differences

  • Sample storage duration

  • Cell cycle stage and differentiation status

In a research example, PIH1D3 mutations in patients showed variable effects on protein expression and dynein arm assembly. Patient PCD12 II:1 (p.Glu43*) showed faint PIH1D3 staining and less severe ODA loss than other patients, while patient GVA30 II:1 (p.Ile164Leufs*11) showed undetectable PIH1D3 and nearly complete ODA/IDA loss . This variability illustrates how different mutations can affect protein levels and function to different degrees, even within the same gene.

What are common pitfalls in PIH1D3 immunofluorescence experiments and their solutions?

When working with PIH1D3 antibodies in immunofluorescence, researchers should be aware of these challenges:

• High background signal:

  • Cause: Insufficient blocking or antibody concentration too high

  • Solution: Extend blocking time (2+ hours), use 5% BSA or 10% normal serum, and optimize antibody dilution (start with 1:250-1:500)

• Weak or absent signal:

  • Cause: Epitope masking during fixation or low PIH1D3 expression

  • Solution: Try different fixation methods (4% PFA vs. methanol) and optimize antigen retrieval (pH 9.0 TE buffer recommended)

• Nonspecific nuclear staining:

  • Cause: Antibody cross-reactivity with nuclear proteins

  • Solution: Pre-absorb antibody with nuclear extract, use more stringent washing, or try alternative antibody clones

• Inconsistent staining between ciliated cells:

  • Cause: Variable PIH1D3 expression or cell cycle differences

  • Solution: Synchronize cultures and ensure consistent differentiation of ciliated cells

• Mislocalization artifacts:

  • Cause: Overexpression of tagged proteins or fixation artifacts

  • Solution: Use antibodies against endogenous protein and compare multiple fixation protocols

Reference protocol for reliable PIH1D3 immunofluorescence:

• Fix samples in 4% PFA for 10 minutes at room temperature

• Permeabilize with 0.1% Triton X-100 for 5 minutes

• Block with 5% normal goat serum for 1-2 hours

• Incubate with primary antibody at 1:250-1:500 dilution overnight at 4°C

• Wash 3× with PBS + 0.1% Tween-20

• Apply fluorescently-labeled secondary antibody at 1:500 for 1 hour

• Counterstain with DAPI and mount with anti-fade medium

For co-localization studies, include markers for different cellular compartments to correctly interpret PIH1D3 localization: γ-tubulin (basal bodies), acetylated-α-tubulin (axonemes), and nuclear markers .

How can researchers resolve contradictory results between PIH1D3 antibody-based techniques and genetic analyses?

When antibody results conflict with genetic data, consider these approaches:

• Methodological reconciliation strategies:

  • Confirm antibody specificity: Test on known PIH1D3-null samples

  • Verify genetic findings: Sequence the entire PIH1D3 gene including introns and regulatory regions

  • Consider posttranscriptional regulation: Assess mRNA stability and translation efficiency

  • Check for alternative splicing: Use RT-PCR with primers spanning multiple exons

• Common scenarios and solutions:

  Scenario	Possible Explanation	Resolution Approach
  Genetic mutation detected but protein present	Alternative splicing or start codon usage	Sequence cDNA and analyze all possible transcripts
  No mutation detected but protein absent	Epigenetic silencing or large deletion	Include methylation analysis and CNV detection
  Truncating mutation but full-length protein detected	Antibody cross-reactivity	Use multiple antibodies targeting different regions
  Missense mutation but protein unstable	Protein degradation	Treat with proteasome inhibitors before analysis

• Case study from literature:
  In patient PCD12 II:1 with a nonsense mutation (p.Glu43*), RT-PCR revealed that the mutation caused exon skipping, removing exon 3 containing both the mutation and the normal start codon. This allowed use of an alternative in-frame start codon in exon 4, potentially explaining the faint PIH1D3 staining detected by immunofluorescence despite the presence of a seemingly severe genetic variant .

• Technical verification approaches:

  • Use multiple antibodies targeting different protein regions

  • Employ mass spectrometry to identify the actual protein present

  • Perform transcript analysis using RNA-Seq

  • Consider posttranslational modifications that might affect antibody binding

When faced with contradictory results, remember that both antibody-based and genetic approaches have limitations. Antibodies may detect cross-reactive proteins, while genetic analyses might miss regulatory mutations or structural variants affecting PIH1D3 expression.