ZDHHC3 Antibody

What is ZDHHC3 and what is its primary function in cellular systems?

ZDHHC3 (also known as GODZ, DHHC-3, ZNF373, or DHHC1 protein) is a zinc finger DHHC-type palmitoyltransferase that catalyzes the addition of palmitate onto various protein substrates. In humans, the canonical protein has 299 amino acid residues with a molecular mass of 34.2 kDa . It is primarily localized in the Golgi apparatus and belongs to the DHHC palmitoyltransferase family .

Functionally, ZDHHC3 plays critical roles in:

• Palmitoylation of GABA receptors on their gamma subunits (GABRG1, GABRG2, GABRG3), which regulates synaptic clustering and cell surface stability

• Palmitoylation of glutamate receptors GRIA1 and GRIA2, leading to their retention in the Golgi

• Potential palmitoylation of DLG4, DNAJC5, and SNAP25

The protein undergoes post-translational modifications itself, including palmitoylation and phosphorylation .

Which experimental applications are most reliable for ZDHHC3 antibody usage?

Based on comprehensive antibody validation data, ZDHHC3 antibodies have been successfully employed in multiple applications with varying reliability:

Western blot remains the gold standard application, showing consistent results across multiple studies and antibody sources .

How can researchers confirm ZDHHC3 antibody specificity in their experimental systems?

Confirming antibody specificity is crucial for generating reliable data. For ZDHHC3 antibodies, consider implementing these validation approaches:

• Genetic knockdown/knockout controls: Use shRNA-mediated knockdown of ZDHHC3 (validated sequence: 5'-CCCAAAGGAAATGCCACTAAA-3') as demonstrated in kidney cell carcinoma studies .

• Overexpression controls: Compare signal in wild-type versus ZDHHC3-overexpressing cells. Adenovirus-mediated ectopic expression systems have been validated for hepatocytes .

• Tissue panel validation: Test antibody reactivity across tissues with known differential expression (heart, lung, liver, skeletal muscle, kidney, testis, thymus, small intestine, and leukocytes) .

• Cross-species reactivity testing: Validate against orthologous proteins from mouse, rat, bovine, or other model organisms to ensure specificity .

• Peptide competition assay: Pre-incubate antibody with immunizing peptide to demonstrate signal reduction in target application.

What are the optimal conditions for Western blot detection of ZDHHC3?

For optimal Western blot detection of ZDHHC3 (34.2 kDa), follow these methodological guidelines:

• Sample preparation:

  • Extract proteins using binding buffer containing protein inhibitor cocktail (1:100)

  • Centrifuge at 14,000×g at 4°C for 10 min

  • Determine protein concentration via BCA assay

• SDS-PAGE separation:

  • Load 20-30 μg of protein per lane on 10-12% gels

  • Include β-actin (42 kDa) as loading control

• Transfer conditions:

  • Transfer to PVDF membrane at 100V for 60-90 minutes

  • Block with 5% skim milk at room temperature for 1 hour

• Antibody incubation:

  • Primary antibody: Use at 1:500 dilution (for sc-377378) or according to manufacturer recommendations

  • Incubate overnight at 4°C

  • Secondary antibody: Incubate at room temperature for 1 hour

• Detection:

  • Visualize using chemiluminescent reagents

  • Analyze band intensity with ImageJ software

Common troubleshooting issues include non-specific bands around 25-30 kDa and 40-45 kDa, which can be minimized by using longer blocking times and more stringent washing steps.

How can researchers effectively co-immunoprecipitate ZDHHC3 with its interaction partners?

For successful co-immunoprecipitation (Co-IP) of ZDHHC3 with its interaction partners:

• Cell lysis protocol:

  • Lyse cells with binding buffer containing protein inhibitor cocktail (1:100)

  • Centrifuge at 14,000×g at 4°C for 10 min

  • Determine protein concentration after centrifugation

• Immunoprecipitation procedure:

  • Mix protein samples (500 μg) with 3 μg mouse ZDHHC3 monoclonal antibody (sc-377378)

  • Incubate overnight at 4°C

  • Add 50 μl protein A/G magnetic beads for 2 hours

  • Wash three times with washing buffer to remove unbound proteins

  • Add binding and protein loading buffers (1:4)

  • Heat at 95°C for 5 min before analysis by western blotting

• Validated interaction partners:

  • IRHOM2 (validated in hepatocytes)

  • SLC9A2 (validated in kidney cell lines)

• Domain-specific interactions:

  • The PDZ domain of ZDHHC3 is essential for binding to certain partners like IRHOM2

This protocol has been successfully used to demonstrate ZDHHC3 interactions in multiple experimental contexts.

How is ZDHHC3 expression altered in cancer, and what are the implications for antibody-based detection?

ZDHHC3 expression shows distinctive patterns across different cancer types:

These differential expression patterns have important implications for antibody-based detection:

• Titration requirements: Lower antibody dilutions may be needed for cancers with reduced ZDHHC3 expression (like KIRC)

• Background considerations: In tissues with low ZDHHC3 expression, distinguishing specific signal from background becomes more challenging

• Control selection: Appropriate positive and negative controls should be selected based on the cancer type being studied

• Correlation with clinical parameters: In KIRC, ZDHHC3 expression negatively correlates with tumor stage, providing a potential biomarker application

What techniques can be used to study ZDHHC3-mediated protein palmitoylation in disease models?

To investigate ZDHHC3-mediated protein palmitoylation in disease contexts:

• Acyl-biotinyl exchange assay:

  • Detects S-palmitoylation of target proteins

  • Has been validated for detecting ZDHHC3-mediated palmitoylation of SLC9A2 in kidney cells

  • Allows quantification of palmitoylation levels with and without ZDHHC3 manipulation

• Metabolic labeling with alkyne-palmitate (Alk16):

  • Incubate cells with Alk16 as a metabolic palmitate analog

  • Visualize palmitoylated proteins via click chemistry

  • Treatment with hydroxylamine (NH₂OH) can confirm thioester-specific palmitoylation

• Fractionation analysis:

  • Separate membrane and nuclear components

  • Allows determination of how ZDHHC3-mediated palmitoylation affects subcellular localization of substrates

  • Has been used to show ZDHHC3 palmitoylation increases IRHOM2 localization to plasma membrane

• Site-directed mutagenesis:

  • Generate palmitoylation-deficient mutants (e.g., cysteine to alanine mutations)

  • The C476A mutation in IRHOM2 has been shown to decrease co-expression with ZDHHC3

  • Allows functional assessment of palmitoylation on protein function

• In vivo models:

  • Hepatocyte-specific ZDHHC3 knockout or overexpression mouse models have been developed

  • Allow assessment of palmitoylation effects in complex disease states like NASH

How does ZDHHC3 tyrosine phosphorylation affect its function and detection by antibodies?

ZDHHC3 undergoes tyrosine phosphorylation, which can significantly impact:

• Enzymatic activity: Phosphorylation can modulate ZDHHC3's palmitoyltransferase activity

• Protein-protein interactions: May affect binding to substrate proteins

• Subcellular localization: Can influence trafficking between cellular compartments

• Epitope masking: Phosphorylation can alter antibody recognition, particularly for antibodies targeting regions containing or adjacent to phosphorylation sites

When studying phosphorylated ZDHHC3:

• Consider using phospho-specific antibodies when available

• For detection of total ZDHHC3 regardless of phosphorylation status, select antibodies targeting regions distal to known phosphorylation sites

• Validate detection using phosphatase treatment controls to ensure consistent recognition

• For co-immunoprecipitation studies, be aware that phosphorylation status may affect interaction partner binding

Research has shown that ZDHHC3 tyrosine phosphorylation can be assessed using anti-phosphotyrosine antibodies after immunoprecipitation with ZDHHC3-specific antibodies .

How can researchers distinguish between ZDHHC3 and other DHHC family members in experimental systems?

The DHHC family contains 23 members with similar domains, making specific detection challenging. To ensure ZDHHC3 specificity:

• Antibody selection strategies:

  • Choose antibodies targeting unique regions outside the conserved DHHC domain

  • Validate against recombinant ZDHHC proteins, particularly closely related members like ZDHHC7

  • Consider using multiple antibodies targeting different epitopes

• Expression pattern analysis:

  • ZDHHC3 is widely expressed with highest levels in heart, lung, liver, skeletal muscle, kidney, testis, thymus, small intestine, and leukocytes

  • Compare expression patterns with other DHHC family members

• Knockout validation:

  • Studies have shown that only ZDHHC3 knockout (not other ZDHHC family members) affects certain target proteins like IRHOM2

  • Generate ZDHHC3-specific knockouts to validate antibody specificity

• Substrate specificity:

  • ZDHHC3 has substrate preferences distinct from other family members

  • Assess known substrate palmitoylation (GABA receptors, glutamate receptors) as functional validation

• Domain-specific interactions:

  • The PDZ domain of ZDHHC3 mediates specific protein interactions

  • Target this domain for selective detection or functional studies

What are the emerging roles of ZDHHC3 in immune cell function and how can researchers study these interactions?

Recent research has revealed important connections between ZDHHC3 and immune function:

• Immune cell infiltration correlations:

  • ZDHHC3 expression positively correlates with infiltration of CD4+ T cells, CD8+ T cells, macrophages, neutrophils, and dendritic cells in KIRC

  • These correlations suggest immunomodulatory roles

• Immune signaling pathways:

  • ZDHHC3 expression is associated with genes involved in:

    • T cell receptor signaling

    • Natural killer cell-mediated cytotoxicity

    • Primary immunodeficiency

    • Intestinal immune network for IgA production

• Methodological approaches to study immune interactions:

  • Single-cell RNA sequencing: Has been used to analyze ZDHHC3 expression in specific immune cell populations

  • Gene Set Enrichment Analysis (GSEA): Revealed association between ZDHHC3 and immune response pathways

  • Weighted Gene Co-expression Network Analysis (WGCNA): Identified ZDHHC3-related gene networks involved in immune regulation

  • Flow cytometry with ZDHHC3 antibodies: Can detect expression in specific immune cell subsets

  • Immune cell infiltration analysis: Using tools like TIMER database to correlate ZDHHC3 expression with immune cell populations

This emerging field offers opportunities to explore ZDHHC3's role in the tumor microenvironment and immunotherapy responses.

What are the common pitfalls in ZDHHC3 antibody usage and how can they be addressed?

Researchers frequently encounter these challenges when using ZDHHC3 antibodies:

• Cross-reactivity with other DHHC family members:

  • Solution: Use antibodies targeting unique regions of ZDHHC3

  • Validate specificity using ZDHHC3 knockout/knockdown controls

  • Consider using multiple antibodies targeting different epitopes

• Variable detection of post-translational modifications:

  • Solution: Be aware that palmitoylation and phosphorylation of ZDHHC3 itself may affect antibody detection

  • Use appropriate controls (phosphatase treatment, palmitoylation inhibitors)

  • Select antibodies with epitopes less affected by modifications

• Inconsistent immunohistochemistry results:

  • Solution: Optimize antigen retrieval methods (heat-induced epitope retrieval at pH 6.0 often works well)

  • Use dilution ranges of 1:200-400 for IHC-P applications

  • Include positive control tissues with known ZDHHC3 expression (heart, lung, liver)

• Weak signal in immunoprecipitation:

  • Solution: Use higher protein amounts (500 μg recommended)

  • Extend antibody incubation time (overnight at 4°C)

  • Use monoclonal antibodies (like sc-377378) that have been validated for IP

• Issues with subcellular localization studies:

  • Solution: Use fractionation controls to verify compartment separation

  • Compare results with known ZDHHC3 localization pattern (primarily Golgi)

  • Co-staining with organelle markers can improve localization accuracy

How can researchers optimize ZDHHC3 antibody usage for detecting low abundance proteins in complex tissues?

For detecting low levels of ZDHHC3 or its substrates in complex tissues:

• Signal amplification strategies:

  • Employ tyramide signal amplification (TSA) for immunohistochemistry/immunofluorescence

  • Use high-sensitivity ECL substrates for Western blotting

  • Consider biotin-streptavidin amplification systems

• Sample enrichment techniques:

  • Perform subcellular fractionation to concentrate Golgi membranes where ZDHHC3 is primarily located

  • Use immunoprecipitation to concentrate ZDHHC3 or its substrates before detection

  • Employ tissue microdissection to isolate relevant regions in heterogeneous samples

• Reducing background strategies:

  • Increase blocking time and concentration (5% BSA or milk for 2+ hours)

  • Use detergent optimization in wash buffers (0.1-0.3% Tween-20)

  • Consider specialized blocking reagents for tissue-specific applications

• Antibody selection considerations:

  • For low abundance detection, monoclonal antibodies often provide better signal-to-noise ratios

  • Affinity-purified polyclonal antibodies can offer improved sensitivity

  • Match antibody sensitivity to application needs (high affinity antibodies for detection, moderate affinity for IP)

• Technical considerations for specific applications:

  • For Western blot: Transfer proteins at lower voltage for longer time to improve transfer efficiency

  • For IHC/IF: Extend primary antibody incubation to overnight at 4°C

  • For ELISA: Use sandwich ELISA format with two different antibodies recognizing different epitopes

These optimization strategies have been successfully employed to detect ZDHHC3 in complex disease models like KIRC and NASH.

How are ZDHHC3 antibodies being utilized in novel therapeutic target identification for cancer and metabolic diseases?

ZDHHC3 antibodies are enabling several innovative research directions for therapeutic development:

• ZDHHC3-SLC9A2 axis in kidney cancer:

  • ZDHHC3 suppression decreases S-palmitoylation of SLC9A2

  • This inhibits apoptosis in kidney cancer cells

  • Antibodies are being used to track this pathway in patient samples and preclinical models

• ZDHHC3-IRHOM2 pathway in nonalcoholic steatohepatitis (NASH):

  • ZDHHC3 mediates S-palmitoylation of IRHOM2

  • Palmitoylated IRHOM2 facilitates membrane localization and accumulation

  • Excessive IRHOM2 abundance drives NASH progression

  • Antibodies against ZDHHC3 are critical for monitoring this pathway

• Prognostic biomarker development:

  • ZDHHC3 expression shows significant correlation with patient outcomes in KIRC

  • Higher expression associates with better prognosis

  • Antibody-based detection methods are being standardized for potential clinical application

• Therapeutic target validation:

  • Hepatocyte-specific ZDHHC3 knockout mice show protection against NASH

  • This suggests ZDHHC3 inhibition could have therapeutic potential

  • Antibodies are being used to validate target engagement in drug development pipelines

• Immune microenvironment modulation:

  • ZDHHC3 correlates with immune cell infiltration in tumors

  • This may influence immunotherapy responses

  • Multiplex immunohistochemistry with ZDHHC3 antibodies is being used to study these relationships

These emerging applications highlight the growing importance of specific and well-validated ZDHHC3 antibodies in translational research.

What are the latest methodological advances combining ZDHHC3 antibodies with other techniques for studying protein palmitoylation networks?

Recent methodological innovations have significantly expanded our ability to study ZDHHC3-mediated palmitoylation:

• Proximity labeling combined with immunoprecipitation:

  • BioID or APEX2 fusion to ZDHHC3 to identify proximal proteins

  • ZDHHC3 antibodies then used to validate interactions via co-IP

  • This approach helps identify the complete "palmitome" regulated by ZDHHC3

• Mass spectrometry-based palmitoylation site mapping:

  • Acyl-RAC (resin-assisted capture) or acyl-biotin exchange (ABE) to enrich palmitoylated proteins

  • ZDHHC3 antibodies used to confirm enzyme-substrate relationships

  • LC-MS/MS analysis identifies specific modified cysteine residues

  • Has been used to confirm palmitoylation of SLC9A2 by ZDHHC3

• Live-cell imaging of palmitoylation dynamics:

  • Fluorescent protein-tagged ZDHHC3 combined with antibody-based detection of substrates

  • Allows temporal and spatial resolution of palmitoylation events

  • FRET-based sensors to monitor protein-protein interactions in real-time

• CRISPR-based genetic screens combined with antibody validation:

  • Genome-wide or targeted CRISPR screens to identify ZDHHC3 substrates

  • ZDHHC3 antibodies used to validate hits via biochemical approaches

  • This systematic approach identifies new substrates and biological functions

• Single-cell analysis of ZDHHC3 expression and activity:

  • Single-cell RNA-seq combined with antibody-based protein detection

  • Reveals heterogeneity in ZDHHC3 expression across cell populations

  • Has been used to confirm ZDHHC3 downregulation in KIRC at single-cell level