FKBP4 Antibody, HRP conjugated

What is FKBP4 and what are its primary cellular functions?

FKBP4 (FK506 Binding Protein 4, 59kDa), also known as FKBP52, is an HSP90-associated co-chaperone protein that plays multiple roles in cellular function. FKBP4 regulates the activity of several client proteins including steroid hormone receptors (androgen, glucocorticoid, progesterone, mineralocorticoid, and estrogen receptors) . It interacts with nuclear factors like NF-kB and other proteins such as Argonaute 2 (AGO2) or Tau . Recent research has identified FKBP4 as a novel PI3K-Akt-mTOR proximal interacting protein, suggesting its involvement in this critical signaling pathway . In neuronal systems, FKBP4 may play a role in the autophagy-lysosomal protein degradation system, particularly important in neurodegenerative contexts .

The protein has a molecular weight of approximately 52 kDa, with some observed variations (51-52 kDa) depending on detection methods . Its subcellular localization spans the cytoplasm (cytosol and cytoskeleton) and nucleus, allowing it to participate in diverse cellular processes . The PPIase activity is primarily attributed to the first PPIase FKBP-type domain (amino acids 1-138), while the C-terminal region (amino acids 375-458) functions to prevent tubulin polymerization .

How does the structure of FKBP4 relate to its functional domains?

FKBP4's functional versatility stems from its multi-domain structure. The protein contains several distinct regions that contribute to its various cellular activities. The N-terminal region houses the PPIase (peptidyl-prolyl isomerase) domain responsible for protein folding assistance . This domain catalyzes the cis-trans isomerization of peptidyl-prolyl bonds, which is crucial for proper protein conformation and function .

The central portion of FKBP4 contains TPR (tetratricopeptide repeat) domains that mediate protein-protein interactions, particularly with HSP90 . These repeats are essential for mitochondrial localization and play a significant role in the protein's chaperone activities . The chaperone activity of FKBP4 primarily resides in the C-terminal region, mainly between amino acids 264 and 400 .

Post-translational modifications further regulate FKBP4's function. Phosphorylation by CK2 results in loss of HSP90 binding activity, providing a regulatory mechanism for modulating chaperone function . Additionally, interaction with S100A1 and S100A2 (but not with S100A6) leads to inhibition of FKBP4-HSP90 interaction, revealing another layer of regulation .

What detection methods are available for FKBP4 and what are their research applications?

FKBP4 can be detected using various immunological and molecular biology techniques, each offering distinct advantages for specific research questions:

• Western Blotting (WB): Allows quantification of FKBP4 protein levels in cell or tissue lysates. HRP-conjugated FKBP4 antibodies provide direct detection without secondary antibodies, streamlining the procedure . Typical dilutions range from 1:500-1:2000, and the protein is detected at approximately 52 kDa .

• Immunohistochemistry (IHC): Enables visualization of FKBP4 in tissue sections, providing insights into expression patterns and subcellular localization. For IHC applications, antibody dilutions typically range from 1:50-1:200 . In cancer research, IHC has revealed that FKBP4 expression is associated with breast cancer progression and prognosis, particularly in ER-negative breast cancer .

• Immunoprecipitation (IP): Facilitates isolation of FKBP4 and its interacting partners for studying protein complexes and signaling networks . This technique has been instrumental in identifying novel FKBP4 interactions with components of the PI3K-Akt-mTOR pathway .

• Immunofluorescence (IF) and Immunocytochemistry (ICC): Provide detailed subcellular localization information. Studies using these techniques have shown FKBP4 localization changes in neurodegenerative diseases and during proteotoxic stress .

• BirA Proximity-Dependent Biotin Identification: A sophisticated approach used to characterize the in vivo proximal interactome of FKBP4 . This technique involves generating FLAGBirA*-FKBP4 fusion constructs, enabling identification of proteins in close proximity to FKBP4 through mass spectrometry .

Each technique requires specific optimization steps and has particular strengths for addressing different research questions about FKBP4 function and interactions.

What factors should be considered when selecting an FKBP4 antibody for specific applications?

Selecting the appropriate FKBP4 antibody requires careful consideration of several factors to ensure experimental success:

• Antibody type and specificity:

  • Monoclonal antibodies (like the Hi52C clone) offer high specificity for a single epitope, providing consistent results across experiments .

  • Polyclonal antibodies recognize multiple epitopes and may provide stronger signals but potentially less specificity .

  • Consider the specific epitope targeted (some antibodies target specific regions such as AA 1-459, AA 301-410, or AA 220-459) .

• Host species and cross-reactivity:

  • Select antibodies raised in species different from your experimental samples to avoid background issues.

  • Review documented cross-reactivity with species of interest. Some FKBP4 antibodies demonstrate reactivity with human, mouse, rat, dog, and hamster proteins .

  • For comparative studies across species, ensure the antibody recognizes conserved epitopes.

• Conjugation and detection system:

  • HRP-conjugated antibodies eliminate the need for secondary antibodies in WB and IHC, reducing background and protocol complexity .

  • Consider the detection system compatibility with your instrumentation and sensitivity requirements.

  • For multiplex applications, select antibodies with appropriate conjugates that allow signal separation.

• Validation for specific applications:

  • Verify that the antibody has been validated for your intended application (WB, IHC, IP, IF, ICC) .

  • Review published literature using the specific antibody for similar experimental systems.

  • Consider performing validation experiments such as peptide competition or knockdown controls.

• Technical specifications:

  • Note the recommended dilution ranges (1:500-1:2000 for WB, 1:50-1:200 for IHC) .

  • Consider the antibody format (liquid vs. lyophilized) and storage requirements.

  • Review stability information and reconstitution protocols if applicable.

Antibody selection should be tailored to the specific research question, experimental system, and intended application to maximize specificity and sensitivity.

How should researchers optimize Western blot protocols for FKBP4 detection using HRP-conjugated antibodies?

Optimizing Western blot protocols for FKBP4 detection requires attention to several critical parameters:

• Sample preparation:

  • Include protease inhibitors in lysis buffers to prevent FKBP4 degradation.

  • For phosphorylation studies, add phosphatase inhibitors to preserve modification states.

  • Maintain consistent protein concentrations across samples (20-50 μg total protein per lane is typically sufficient).

  • Denature samples completely (95°C for 5 minutes) to ensure proper antibody access to epitopes.

• Gel electrophoresis considerations:

  • Use 10-12% acrylamide gels for optimal resolution around 52 kDa, the expected molecular weight of FKBP4 .

  • Include molecular weight markers that bracket the expected 52 kDa band.

  • Be aware that heavy chain from immunoprecipitation samples migrates close to FKBP4 on SDS-PAGE, which may complicate interpretation .

• Transfer and blocking parameters:

  • For proteins around 52 kDa, standard transfer conditions (100V for 60-90 minutes) are generally sufficient.

  • Block membranes thoroughly (5% BSA or non-fat milk in TBST for 1 hour at room temperature).

  • For phosphorylation-specific detection, BSA is preferred over milk for blocking.

• Antibody incubation:

  • Dilute HRP-conjugated FKBP4 antibody to manufacturer-recommended concentration (typically 1:500-1:2000) .

  • Incubate overnight at 4°C for maximum sensitivity or 1-2 hours at room temperature for convenience.

  • Perform extensive washing steps (3-5 washes of 5-10 minutes each) to minimize background.

• Detection and visualization:

  • Use enhanced chemiluminescence (ECL) substrate compatible with HRP.

  • Begin with short exposure times (30 seconds) and increase as needed to avoid signal saturation.

  • For quantitative comparisons, ensure signals fall within the linear range of detection.

  • Include appropriate loading controls (β-actin, GAPDH) for normalization.

• Troubleshooting considerations:

  • If multiple bands appear, perform peptide competition assays to identify specific FKBP4 signal.

  • For weak signals, increase protein loading, decrease antibody dilution, or use more sensitive ECL substrates.

  • For high background, increase washing stringency and optimize blocking conditions.

Systematic optimization of these parameters will enhance specificity and sensitivity for FKBP4 detection in Western blot applications.

What are the critical steps for immunohistochemical detection of FKBP4 in tissue samples?

Successful immunohistochemical detection of FKBP4 in tissue samples depends on precise execution of several critical steps:

• Tissue preparation and fixation:

  • For FFPE tissues, standardize fixation time (24-48 hours in 10% neutral buffered formalin) to maintain epitope integrity.

  • For frozen sections, fix briefly (10 minutes in 4% paraformaldehyde) before proceeding with staining.

  • Section thickness (4-6 μm) should be consistent across experimental and control samples.

• Antigen retrieval optimization:

  • Heat-mediated antigen retrieval is typically necessary for FKBP4 detection in FFPE tissues.

  • Test both citrate buffer (pH 6.0) and high pH EDTA buffer (pH 9.0) to determine optimal conditions .

  • Standardize retrieval time and temperature (typically 20 minutes at 95-98°C) for consistent results.

• Blocking procedures:

  • Quench endogenous peroxidase activity (3% H₂O₂, 10 minutes) before antibody incubation .

  • Block non-specific binding sites with appropriate blocking reagents (EnVision™ FLEX or similar) .

  • Consider additional blocking steps for tissues with high background (avidin/biotin block if using biotin-based systems).

• Antibody incubation and dilution:

  • Optimize antibody dilution through titration experiments (typically 1:50-1:200 for IHC) .

  • Standardize incubation time and temperature (20 minutes at room temperature or overnight at 4°C) .

  • Include positive control tissues known to express FKBP4 and negative controls (antibody diluent without primary antibody).

• Detection system:

  • For HRP-conjugated antibodies, use diaminobenzidine (DAB) as a chromogen .

  • Standardize development time (3-5 minutes) for consistent signal intensity.

  • Counterstain with hematoxylin for nuclear visualization and context .

• Scoring and quantification:

  • Implement a standardized scoring system combining signal intensity (0=none, 1=mild, 2=moderate, 3=intense) and percentage of positive cells (0-100%) .

  • Convert raw scores to categorical classifications (low, medium, high) for statistical analysis .

  • Consider digital image analysis for objective quantification.

• Validation approaches:

  • Compare FKBP4 staining patterns with published literature.

  • Include tissues with known differential expression of FKBP4 (e.g., normal breast vs. breast cancer) .

  • Perform parallel staining with different FKBP4 antibodies targeting distinct epitopes.

Attention to these methodological details ensures reproducible and reliable FKBP4 detection in tissue specimens.

How does FKBP4 function in cancer progression and what methodologies best reveal its role?

FKBP4 plays significant roles in cancer biology, particularly in breast cancer progression. Research has revealed several methodological approaches to investigate its functions:

• Expression analysis in cancer vs. normal tissues:

  • IHC studies using HRP-conjugated FKBP4 antibodies have shown that FKBP4 is upregulated in breast cancer tissues compared to normal controls .

  • This upregulation appears particularly significant in estrogen receptor-positive tissues/cells, suggesting hormone-dependent regulation .

  • Western blot analysis of cancer cell lines corroborates these findings at the protein level .

• Functional studies through genetic manipulation:

  • FKBP4 depletion specifically reduces cell growth and proliferation in triple-negative breast cancer cell models .

  • Xenograft tumor models with FKBP4 knockdown show reduced tumor growth, providing in vivo validation of its pro-tumorigenic role .

  • These findings suggest FKBP4 as a potential therapeutic target, particularly in hormone receptor-negative cancers with limited treatment options.

• Mechanistic investigations:

  • Protein interactome strategies using BirA proximity-dependent biotin identification have demonstrated that FKBP4 is a novel PI3K-Akt-mTOR proximal interacting protein .

  • This interaction facilitates Akt phosphorylation through PI3K/PDK1 and mTORC2, promoting cell growth and proliferation .

  • Co-immunoprecipitation studies confirm these protein-protein interactions and help elucidate the molecular mechanisms underlying FKBP4's role in cancer.

• Prognostic correlation studies:

  • Analysis of FKBP4 expression levels correlates with breast cancer progression and prognosis, particularly in ER-negative breast cancer .

  • Tissue microarray studies comparing FKBP4 expression across tumor stages can reveal its utility as a prognostic biomarker.

  • Kaplan-Meier survival analysis stratified by FKBP4 expression levels provides clinical relevance to these findings.

• Therapeutic response prediction:

  • Cell viability assays following FKBP4 modulation can predict sensitivity to conventional chemotherapeutics.

  • Combining FKBP4 inhibition with targeted therapies may reveal synergistic effects worth pursuing clinically.

  • Patient-derived xenograft models with varying FKBP4 expression can evaluate treatment response heterogeneity.

These methodological approaches collectively build a comprehensive understanding of FKBP4's role in cancer biology and highlight its potential as a therapeutic target or biomarker.

What methods are most effective for investigating FKBP4's role in neurodegenerative diseases?

FKBP4's emerging role in neurodegenerative diseases requires specialized methodological approaches to elucidate its functions:

• Expression analysis in neural tissues:

  • IHC studies have revealed that FKBP4 protein expression is strongly reduced in the frontal cortex of Alzheimer's disease (AD) and frontotemporal lobar degeneration with Tau (FTLD-Tau) .

  • Western blot quantification can provide more precise measurements of this reduction across brain regions.

  • Single-cell RNA sequencing can identify cell-type-specific changes in FKBP4 expression in disease states.

• Subcellular localization studies:

  • Immunofluorescence microscopy shows FKBP4 localization in the lysosomal system of healthy human neurons, suggesting its role in protein degradation pathways .

  • In pathological conditions, FKBP4 shows altered localization patterns, including perinuclear clustering around lysosomes .

  • Co-localization studies with MAPT/Tau and MAP1LC3/LC3-positive autophagic vesicles reveal functional associations during proteotoxic stress .

• Functional studies in neuronal models:

  • FKBP4 knockdown in SH-SY5Y human neuronal cells affects autophagy-lysosomal system function under MAPT-induced proteotoxic stress .

  • Studies in dorsal root ganglion (DRG) neurons from human MAPT P301S transgenic mice demonstrate that FKBP4 deficiency decreases MAP1LC3-II expression and provokes MAPT accumulation during long-term stress .

  • Live-cell imaging with fluorescently tagged FKBP4 can track dynamic changes in localization during stress responses.

• Autophagy and protein degradation assays:

  • FKBP4 decrease alters lysosomal clustering and increases MAPT and MAP1LC3 secretion in neuronal models .

  • Autophagy flux assays (monitoring LC3-II turnover in the presence/absence of lysosomal inhibitors) can measure how FKBP4 levels affect autophagy efficiency.

  • Proximity ligation assays can detect in situ interactions between FKBP4 and autophagy components.

• Rescue experiments:

  • Ectopic FKBP4 expression can prevent MAPT secretion after MAPT accumulation in neuronal cells, suggesting a regulatory role in proteostasis .

  • Dose-response studies can determine threshold levels of FKBP4 required for maintaining normal autophagy function.

  • Structure-function analyses using FKBP4 mutants can identify domains crucial for its neuroprotective effects.

These methodological approaches collectively provide insights into how FKBP4 dysfunction may contribute to neurodegenerative pathology through impaired protein degradation and increased toxic protein accumulation.

How can researchers effectively study the interaction between FKBP4 and the PI3K-Akt-mTOR signaling pathway?

The interaction between FKBP4 and the PI3K-Akt-mTOR pathway represents a significant area of research with implications for both cancer and neurodegenerative diseases. Effective methodological approaches include:

• Protein interaction mapping:

  • BirA proximity-dependent biotin identification represents a cutting-edge approach for characterizing the in vivo proximal interactome of FKBP4 .

  • This technique involves generating FLAGBirA*-FKBP4 fusion constructs, expressing them in appropriate cell systems, and identifying biotinylated proteins through mass spectrometry .

  • Co-immunoprecipitation studies using HRP-conjugated or unconjugated FKBP4 antibodies can validate direct interactions with pathway components like PI3K, PDK1, and mTORC2 .

• Signaling cascade analysis:

  • Western blot analysis of phosphorylated Akt (at Ser473 and Thr308) in FKBP4-depleted versus control cells can demonstrate FKBP4's effect on pathway activation .

  • Time-course experiments following growth factor stimulation can reveal how FKBP4 influences signaling kinetics.

  • Pharmacological inhibition of specific pathway components (PI3K, mTOR) can help position FKBP4 within the signaling cascade.

• Functional outcome measurements:

  • Cell proliferation assays in models with modulated FKBP4 expression can connect pathway alterations to biological outcomes .

  • Glucose metabolism measurements (uptake assays, lactate production) can assess metabolic consequences of FKBP4-mediated pathway modulation.

  • Protein synthesis assays (puromycin incorporation, polysome profiling) can evaluate translational effects downstream of mTORC1.

• Subcellular localization studies:

  • Immunofluorescence microscopy to track co-localization of FKBP4 with pathway components under different stimulation conditions.

  • Subcellular fractionation followed by Western blotting to quantify compartment-specific interactions.

  • Live-cell imaging with fluorescently tagged components to observe dynamic assembly of signaling complexes.

• Genetic manipulation approaches:

  • CRISPR/Cas9-mediated knockout of FKBP4 followed by rescue experiments with wild-type or mutant constructs.

  • Domain-specific mutations to identify regions of FKBP4 critical for pathway interaction.

  • Inducible expression systems to study acute versus chronic effects of FKBP4 modulation on pathway activity.

• In vivo validation:

  • Tissue-specific knockout models to study pathway alterations in physiologically relevant contexts.

  • Xenograft models with FKBP4-modulated cells treated with pathway inhibitors to assess therapeutic implications.

  • Analysis of patient samples for correlations between FKBP4 expression and pathway activation markers.

These methodological approaches collectively build a comprehensive understanding of how FKBP4 interfaces with the PI3K-Akt-mTOR pathway and influences downstream biological processes.

What are the critical steps for validating FKBP4 antibody specificity in experimental systems?

Validating antibody specificity is crucial for generating reliable data with FKBP4 antibodies. Researchers should implement these rigorous validation approaches:

• Peptide competition assays:

  • Pre-incubate the FKBP4 antibody with excess immunizing peptide before application to samples.

  • A specific signal should be significantly reduced or eliminated after peptide competition .

  • Include a non-specific peptide control to confirm specificity of competition.

  • Document results with side-by-side images or quantitative signal measurements.

• Genetic knockdown/knockout validation:

  • Generate FKBP4 knockdown (siRNA/shRNA) or knockout (CRISPR/Cas9) cell lines.

  • Compare antibody signals between wild-type and FKBP4-depleted samples across multiple applications (WB, IF, IHC).

  • The specific signal should be proportionally reduced or eliminated in knockdown/knockout samples.

  • Include controls for off-target effects by rescuing with exogenous FKBP4 expression.

• Multiple antibody approach:

  • Test multiple FKBP4 antibodies targeting different epitopes (N-terminal, central region, C-terminal).

  • Consistent patterns across different antibodies increase confidence in specificity.

  • Document convergent and divergent results across antibody types.

  • Consider both monoclonal and polyclonal antibodies in this comparative approach.

• Signal verification by molecular weight:

  • For Western blotting, verify that the detected band appears at the expected molecular weight (~52 kDa) .

  • Be aware that post-translational modifications may cause slight shifts in migration patterns.

  • Note that heavy chains from immunoprecipitation experiments migrate close to FKBP4 on SDS-PAGE, which may complicate interpretation .

  • Use molecular weight markers that bracket the expected FKBP4 size.

• Cross-reactivity assessment:

  • Test the antibody against recombinant FKBP family proteins (particularly FKBP5/FKBP51, which shares structural similarities with FKBP4).

  • Evaluate performance in species known to express FKBP4 homologs with varying sequence conservation.

  • Document cross-reactivity with dog, hamster, human, mouse, and rat samples as appropriate for your research .

• Mass spectrometry validation:

  • Perform immunoprecipitation with the FKBP4 antibody followed by mass spectrometry analysis.

  • Confirm that FKBP4 is among the most abundant proteins in the precipitate.

  • Identify any non-specific binding partners for future reference.

These validation approaches should be documented in laboratory records and included in publications to enhance reproducibility and reliability of FKBP4 research.

How can researchers troubleshoot common issues with FKBP4 antibodies in different applications?

Troubleshooting issues with FKBP4 antibodies requires systematic approach to identify and resolve experimental problems:

• Western Blot Issues and Solutions:

  Issue	Potential Causes	Solutions
  No signal	Insufficient protein, improper transfer, inactive antibody	Increase protein loading, verify transfer, test antibody with positive control
  Multiple bands	Degradation, splice variants, cross-reactivity	Include protease inhibitors, verify with peptide competition, optimize antibody dilution
  High background	Insufficient blocking, excessive antibody, inadequate washing	Increase blocking time, dilute antibody further, increase wash stringency
  Heavy chain interference	IP samples showing band at ~50 kDa	Use HRP-conjugated light-chain specific secondary antibodies, note that heavy chain migrates close to FKBP52 on SDS-PAGE

• Immunohistochemistry Troubleshooting:

  Issue	Potential Causes	Solutions
  Weak or absent staining	Overfixation, inadequate retrieval, low antibody concentration	Optimize antigen retrieval, increase antibody concentration, extend incubation time
  Non-specific staining	Insufficient blocking, excessive antibody	Improve blocking, dilute antibody, include additional blocking steps
  Variable staining intensity	Inconsistent fixation, processing variations	Standardize fixation protocols, process all samples simultaneously
  Edge artifacts	Drying during incubation	Maintain humid chamber, use adequate volume of reagents

• Immunofluorescence Challenges:

  Issue	Potential Causes	Solutions
  High background fluorescence	Autofluorescence, non-specific binding	Include quenching steps, optimize blocking, use spectral unmixing
  Weak signal	Low expression, epitope masking, photobleaching	Optimize fixation, try different retrieval methods, use anti-fade mounting media
  Unexpected subcellular pattern	Fixation artifacts, cross-reactivity	Compare patterns with published data, validate with different antibodies
  Inconsistent results	Protocol variations, antibody stability	Standardize all steps, aliquot antibodies to avoid freeze-thaw cycles

• IP-specific considerations:

  • Low IP efficiency may result from epitope masking in protein complexes.

  • Cross-linking approaches can stabilize transient interactions.

  • For co-IP experiments, gentler lysis conditions help maintain protein-protein interactions.

  • Note that some interactions may be cell-type specific or condition-dependent.

• Quantification challenges:

  • For densitometry, ensure signals fall within linear range of detection.

  • When comparing expression across samples, include appropriate loading controls.

  • For IHC quantification, standardize scoring methods and involve multiple independent scorers.

  • Consider digital image analysis with standardized parameters for objective quantification.

• Antibody storage and handling:

  • Maintain proper storage conditions (-20°C for long-term, 4°C for working aliquots).

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots.

  • Include carrier proteins (BSA) and preservatives for diluted antibodies.

  • Monitor expiration dates and performance over time.

Systematic troubleshooting following these guidelines will help researchers optimize FKBP4 antibody performance across different applications.

What are the best practices for quantitative analysis of FKBP4 expression across different experimental systems?

Accurate quantitative analysis of FKBP4 expression requires rigorous methodological approaches to ensure reliability and reproducibility:

• Western Blot Quantification:

  • Use gradient or precast gels with consistent composition for reproducible protein separation.

  • Include standard curves with recombinant FKBP4 protein to verify linear detection range.

  • Apply multiple loading controls (β-actin, GAPDH, total protein staining) to normalize for loading variations.

  • Capture images using digital systems with broad dynamic range rather than film.

  • Perform densitometry using software that measures integrated density within linear range.

  • Express results as relative rather than absolute values when comparing across experiments.

• Immunohistochemistry Quantification:

  • Implement a standardized scoring system combining intensity (0-3+) and percentage of positive cells (0-100%) .

  • Use digital pathology platforms for objective assessment when possible.

  • Employ color deconvolution algorithms to separate DAB signal from hematoxylin counterstain.

  • Analyze multiple fields per sample (minimum 3-5) to account for tissue heterogeneity.

  • Include positive and negative control tissues in each batch to normalize for staining variations.

  • Have multiple observers score samples independently to minimize subjective bias.

• RT-qPCR Analysis of FKBP4 mRNA:

  • Select multiple reference genes validated for stability in your experimental system.

  • Verify primer efficiency (90-110%) using standard curves.

  • Use the comparative Ct (2^-ΔΔCt) method with appropriate normalization.

  • Include no-template and no-RT controls in each experiment.

  • Perform technical triplicates and biological replicates for statistical validity.

  • Validate significant expression changes at the protein level.

• Statistical Analysis Considerations:

  • Determine appropriate statistical tests based on data distribution and experimental design.

  • Perform power analysis to ensure adequate sample size.

  • Apply appropriate corrections for multiple comparisons.

  • Report effect sizes alongside p-values for better interpretation of biological significance.

  • Consider non-parametric tests when assumptions of normality cannot be met.

• Cross-platform Validation:

  • Confirm expression changes using orthogonal techniques (e.g., validate WB findings with IHC).

  • Compare protein expression with mRNA levels to identify post-transcriptional regulation.

  • When comparing results across studies, consider methodological differences in antibodies, sample preparation, and quantification approaches.

• Reporting Standards:

  • Document detailed methodology including antibody catalog numbers, dilutions, incubation conditions, and image acquisition parameters.

  • Provide representative images showing the full range of expression patterns observed.

  • Include all quantification methods, normalization strategies, and statistical approaches used.

  • Present raw data alongside processed results when possible.

Following these best practices ensures robust quantitative analysis of FKBP4 expression across different experimental systems and facilitates comparison between studies.

How can researchers utilize FKBP4 antibodies to investigate hormone receptor signaling mechanisms?

FKBP4's role as a co-chaperone for steroid hormone receptors makes it a critical factor in hormone signaling research. Effective methodological approaches include:

• Receptor-FKBP4 interaction studies:

  • Co-immunoprecipitation using HRP-conjugated or unconjugated FKBP4 antibodies can reveal physical interactions with steroid hormone receptors (ER, PR, AR, GR, MR) .

  • Proximity ligation assays provide in situ visualization of these interactions at the single-cell level.

  • FRET or BRET approaches can measure dynamic interactions in living cells.

  • BirA proximity labeling can identify the broader complex of proteins associated with FKBP4-receptor interactions.

• Functional impact assessment:

  • Luciferase reporter assays with hormone-responsive elements can measure how FKBP4 modulation affects receptor transcriptional activity.

  • ChIP-seq analysis following FKBP4 knockdown can identify genome-wide changes in receptor binding patterns.

  • RNA-seq can reveal how FKBP4 alterations affect the broader transcriptional landscape downstream of receptor activation.

  • Hormone binding assays can determine whether FKBP4 affects receptor-ligand affinity.

• Nuclear translocation dynamics:

  • Immunofluorescence time-course experiments can track receptor localization following hormone stimulation in FKBP4-modulated cells.

  • High-content imaging with automated quantification provides statistical power for detecting subtle changes.

  • FKBP4 interaction with dynein may affect nuclear receptor transport, which can be investigated using cytoskeletal inhibitors .

  • Subcellular fractionation followed by Western blotting provides biochemical verification of translocation efficiency.

• Post-translational modification analysis:

  • Phospho-specific antibodies can determine how FKBP4 affects receptor phosphorylation status.

  • Mass spectrometry approaches can identify novel modifications regulated by FKBP4.

  • In vitro kinase assays can test direct effects of FKBP4 on receptor modification.

  • Phosphorylation of FKBP4 itself by CK2 results in loss of HSP90 binding activity, providing a regulatory checkpoint .

• Tissue-specific signaling analysis:

  • IHC with FKBP4 antibodies in hormone-responsive tissues can reveal expression patterns correlating with receptor activity.

  • Tissue-specific knockout models can demonstrate context-dependent roles of FKBP4 in hormone signaling.

  • Patient-derived samples can be analyzed for correlations between FKBP4 levels and hormone-dependent disease progression.

These approaches collectively provide comprehensive insights into how FKBP4 regulates hormone receptor signaling, with implications for diseases ranging from cancer to reproductive and metabolic disorders.

How should researchers design experiments to study FKBP4's role in the autophagy-lysosomal system?

FKBP4's emerging role in the autophagy-lysosomal system requires specialized experimental approaches:

• Lysosomal morphology and distribution studies:

  • Confocal microscopy using FKBP4 antibodies co-stained with lysosomal markers (LAMP1, LAMP2) can reveal spatial relationships.

  • Research has shown that acute MAPT accumulation in neuronal cells induces perinuclear clustering of lysosomes and triggers FKBP4 localization around these clusters .

  • Live-cell imaging with fluorescently tagged FKBP4 can track dynamic associations with lysosomes during stress responses.

  • Super-resolution microscopy provides detailed visualization of FKBP4's association with specific lysosomal membrane domains.

• Autophagy flux assessment:

  • Western blot analysis of MAP1LC3-II levels in FKBP4-depleted versus control cells, with and without lysosomal inhibitors (bafilomycin A1, chloroquine) .

  • Tandem fluorescent-tagged LC3 (mRFP-GFP-LC3) assays can distinguish autophagosome formation from fusion with lysosomes.

  • Long-lived protein degradation assays provide functional measurement of autophagy efficiency.

  • Studies in mouse DRG neurons showed that FKBP4 deficiency decreased MAP1LC3-II expression during long-term stress .

• Protein aggregation and clearance experiments:

  • Monitoring MAPT/Tau accumulation in FKBP4-modulated neuronal models under proteotoxic stress conditions .

  • Pulse-chase experiments to measure clearance rates of aggregation-prone proteins.

  • Filter trap assays to quantify insoluble protein aggregates.

  • FKBP4 decrease has been shown to alter lysosomal clustering along with increased MAPT and MAP1LC3 secretion .

• Secretion and exosome analysis:

  • Quantification of extracellular MAPT and MAP1LC3 following FKBP4 depletion .

  • Ultracentrifugation to isolate extracellular vesicles for component analysis.

  • Nanoparticle tracking analysis to characterize exosome size and concentration.

  • Ectopic FKBP4 expression prevented MAPT secretion after MAPT accumulation in SH-SY5Y cells, suggesting a regulatory role .

• Interaction with autophagy machinery:

  • Co-immunoprecipitation studies to identify direct interactions between FKBP4 and autophagy regulators.

  • Proximity labeling approaches to map the broader autophagy-related interactome.

  • In vitro binding assays to determine binding affinities and domains involved.

  • Genetic epistasis experiments combining FKBP4 modulation with alterations in canonical autophagy genes.

• Therapeutic modulation approaches:

  • Screen for compounds that restore normal lysosomal function in FKBP4-deficient cells.

  • Test FKBP4 overexpression as a potential therapeutic strategy in neurodegenerative disease models.

  • Evaluate combinations with established autophagy modulators for synergistic effects.

These experimental designs provide comprehensive assessment of FKBP4's role in autophagy-lysosomal function, with particular relevance to neurodegenerative disease mechanisms.