EXO70A1 Antibody

What is EXO70A1 and why is it important in cell biology research?

EXO70A1 is a conserved subunit of the exocyst complex, an octameric protein assembly that tethers secretory vesicles to the plasma membrane during exocytosis. In plants, EXO70A1 plays a crucial role in conventional exocytosis, polar cell growth, and cell wall biogenesis . Unlike its mammalian counterparts (like EXO70/EXOC7), plant EXO70A1 belongs to a greatly expanded gene family that has evolved specialized functions, making it a valuable subject for studying evolutionary adaptation of fundamental cellular machinery . EXO70A1 is particularly important for developmental processes, as demonstrated by the severe phenotypes in exo70a1 mutants which include reduced hypocotyl elongation and compromised cell wall formation .

What types of EXO70A1 antibodies are currently available for plant research?

Based on the literature, several types of EXO70A1 antibodies have been successfully employed in plant research:

• Polyclonal antibodies raised against specific peptide regions of Arabidopsis EXO70A1

• Antibodies recognizing conserved epitopes that work across multiple plant species

• Anti-tag antibodies (anti-GFP, anti-HA) used with tagged versions of EXO70A1 for immunoprecipitation and localization studies

The type of antibody selected should depend on experimental goals, with polyclonal antibodies generally providing higher sensitivity but potentially lower specificity than monoclonal options.

What are the optimal protocols for using EXO70A1 antibodies in Western blotting of plant samples?

For optimal Western blotting of plant samples with EXO70A1 antibodies:

• Sample preparation: Extract proteins from plant tissues using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, protease inhibitor cocktail, and 1 mM PMSF. Fresh tissue yields better results than frozen samples.

• Protein amount: Load 20-30 μg of total protein per lane with appropriate controls.

• Detection parameters:

  • Primary antibody dilutions: 1:1000 to 1:3000 for commercially available anti-EXO70A1 antibodies

  • Incubation: Overnight at 4°C

  • Secondary antibody: Anti-rabbit HRP at 1:5000 dilution for 1 hour at room temperature

  • Expected band size: Approximately 76 kDa for Arabidopsis EXO70A1

• Controls:

  • Positive control: Wild-type plant extract

  • Negative control: exo70a1 mutant extract (though some truncated protein may be detected in certain mutant alleles)

  • Loading control: Anti-actin antibody on the same membrane

How can EXO70A1 antibodies be optimized for immunoprecipitation studies to investigate exocyst complex interactions?

For successful immunoprecipitation of EXO70A1 and associated exocyst components:

• Pre-clearing step: Incubate plant lysate with protein A/G beads for 1 hour at 4°C to reduce non-specific binding.

• Antibody binding:

  • Use 2-5 μg of anti-EXO70A1 antibody per 500 μg of protein extract

  • Incubate overnight at 4°C with gentle rotation

  • Add pre-washed protein A/G beads and incubate for additional 3-4 hours

• Washing conditions: Perform 4-5 washes with decreasing salt concentrations to maintain specific interactions while removing non-specific binding.

• Validation approach: As demonstrated in multiple studies, co-immunoprecipitation of EXO70A1 should pull down other exocyst subunits such as SEC6, SEC8, and SEC15b that can be detected by mass spectrometry or specific antibodies .

• Critical considerations:

  • Add phosphatase inhibitors if studying phosphorylation-dependent interactions

  • Consider mild crosslinking for capturing transient interactions

  • For detecting interaction partners by mass spectrometry, use a higher starting amount of protein (1-2 mg)

What techniques have proven effective for validating direct interaction between EXO70A1 and its binding partners?

Several complementary techniques have been successfully used to validate EXO70A1 interactions:

• DARTS (Drug Affinity Responsive Target Stability): This approach has been used to test the interaction between small molecules like Endosidin2 (ES2) and EXO70A1. The technique works on the principle that protein-ligand binding can protect against protease degradation .

• Saturation-Transfer Difference NMR (STD-NMR): Successfully employed to demonstrate direct binding between EXO70A1 and small molecules, this technique can detect interactions with affinity in the micromolar range .

• Microscale Thermophoresis (MST): Used to measure binding affinities between EXO70A1 and its ligands with Kd values in the micromolar range (approximately 253 ± 63.5 μM for interaction with ES2) .

• Yeast Two-Hybrid (Y2H) assays: Effective for mapping interaction domains, as demonstrated by truncation studies showing that the ABC- variant of EXO70A1 strongly interacts with SEC3a and EXO84b-N, while the AB– variant interacts only weakly with EXO84b-N .

• In vitro lipid binding assays: Protein-lipid overlay and cosedimentation with large unilamellar vesicles have demonstrated that EXO70A1 binds to phospholipids like PA, PI4P, and PIP2 .

How can immunohistochemistry with EXO70A1 antibodies be optimized for subcellular localization studies?

For optimal immunohistochemistry with EXO70A1 antibodies:

• Fixation protocol:

  • For plant tissues: 4% paraformaldehyde in PBS for 1-2 hours at room temperature or 2% paraformaldehyde with 0.1% glutaraldehyde for better ultrastructural preservation

  • Avoid over-fixation as it can mask epitopes

• Permeabilization:

  • Use 0.1-0.5% Triton X-100 in PBS for 15-30 minutes

  • For thick sections, consider vacuum infiltration to enhance antibody penetration

• Blocking and antibody dilutions:

  • Block with 3-5% BSA or normal serum for 1 hour

  • Primary antibody: 1:100 to 1:250 dilution, incubate overnight at 4°C

  • Secondary antibody: Fluorophore-conjugated (Alexa Fluor 488/594/647) at 1:200-1:400 dilution

• Controls and validation:

  • Negative control: Primary antibody omission and exo70a1 mutant tissue

  • Positive control: Tissues with known high expression (e.g., root tips, developing xylem)

  • Co-localization: Consider dual labeling with markers for plasma membrane or vesicle trafficking

• Imaging parameters:

  • Use confocal microscopy with appropriate resolution for subcellular structures

  • When studying dynamic processes, consider spinning disk confocal for faster acquisition

How can EXO70A1 antibodies be used to investigate functional specialization among EXO70 paralogs?

To investigate functional specialization among EXO70 paralogs:

• Comparative immunoprecipitation:

  • Perform parallel immunoprecipitations with antibodies against different EXO70 paralogs

  • Compare interacting partners through mass spectrometry to identify unique and shared interactions

  • This approach revealed different exocyst subcomplex formations, such as EXO70B1 associating with EXO84b and SEC5a in a complex distinct from EXO70A1-containing complexes

• Paralog-specific expression analysis:

  • Use tissue and cell-type specific analysis to compare expression patterns of different EXO70 paralogs

  • For example, comparing EXO70A1, EXO70B1, and EXO70B2 expression patterns under pathogen challenge revealed that EXO70B2 is significantly upregulated at 4 hours post-inoculation while EXO70A1 levels slightly decreased

• Cross-complementation studies:

  • Express different EXO70 paralogs under the same promoter in mutant backgrounds

  • Analysis revealed that EXO70A1 was functionally substituted only by its closest paralog, EXO70A2, while none of the tested EXO70 isoforms (including A1, A2, B2, C1, D2, F1, and H7) could substitute for EXO70B1

  Paralog Expressed	Can Complement exo70a1?	Can Complement exo70b1?
  EXO70A1	Yes	No
  EXO70A2	Yes	No
  EXO70B1	No	Yes
  EXO70B2	No	No
  EXO70C1	No	No
  EXO70D2	No	No
  EXO70F1	No	No
  EXO70H7	No	No

What approaches can be used to study domain-specific functions of EXO70A1 using antibodies?

Domain-specific functions of EXO70A1 can be studied through:

• Domain truncation combined with antibody detection:

  • Create truncated variants of EXO70A1 lacking specific domains (A, B, C, or D)

  • Express these variants in exo70a1 mutant background

  • Use anti-EXO70A1 antibodies or epitope-tag antibodies to analyze localization and function

  • This approach revealed that while the ABC- variant strongly interacts with SEC3a and EXO84b-N, it fails to localize to the plasma membrane

• Domain-specific antibodies:

  • Develop antibodies targeting specific domains of EXO70A1

  • Use these to study differential exposure of domains in various cellular contexts

  • This could provide insights into conformational changes associated with activation

• Protection/accessibility assays:

  • Similar to DARTS (Drug Affinity Responsive Target Stability), use limited proteolysis with domain-specific antibody protection

  • Areas bound by antibodies will be protected from proteolysis

  • This can reveal which domains are accessible in different cellular contexts or protein states

• Structure-function correlation:

  • Based on the crystal structure of Arabidopsis EXO70A1 at 3.1Å resolution

  • The structure reveals three domains based on interdomain hinge points: N-terminal (75-379), C-terminal (511-629), and middle (380-510)

  • Compare antibody reactivity with structural features to understand domain accessibility

How can EXO70A1 antibodies be used to study alterations in exocyst complex dynamics during stress responses?

To study exocyst complex dynamics during stress responses:

• Quantitative immunoprecipitation:

  • Perform immunoprecipitation with anti-EXO70A1 antibodies from control and stressed plants

  • Quantify co-precipitated exocyst subunits by western blot or mass spectrometry

  • Changes in the stoichiometry of co-precipitated subunits may indicate stress-induced complex remodeling

• Stress-induced localization changes:

  • Use immunofluorescence with anti-EXO70A1 antibodies to track localization changes under stress

  • Examples include pathogen challenge, where studies showed EXO70B2 is specifically upregulated in early hours after fungal attack and accumulates at attack/papillae sites, while EXO70A1 levels slightly decrease

• Phosphorylation state analysis:

  • Immunoprecipitate EXO70A1 from control and stressed plants

  • Analyze phosphorylation status by phospho-specific antibodies or mass spectrometry

  • Changes in phosphorylation may indicate altered regulation under stress

• Comparative recovery after photobleaching:

  • In systems using fluorescently-tagged EXO70A1, measure FRAP (Fluorescence Recovery After Photobleaching) parameters

  • Compare recovery rates between control and stress conditions

  • Validate findings using antibody-based approaches in fixed cells

What experimental approaches can investigate how EXO70A1 recruits other exocyst components to the plasma membrane?

To investigate EXO70A1's role in recruiting exocyst components:

• Sequential immunoprecipitation:

  • First precipitate with anti-EXO70A1 antibodies

  • Elute under mild conditions

  • Perform second immunoprecipitation with antibodies against other exocyst subunits

  • This can identify subcomplexes and assembly intermediates

• In vitro reconstitution assays:

  • Purify recombinant EXO70A1 and other exocyst components

  • Use fluorescently labeled artificial membranes containing specific phospholipids

  • Add EXO70A1 followed by other components to observe sequential recruitment

  • This approach revealed that EXO70A1 binds to phospholipids PA, PI4P, and PIP2 with 80-90% of the input protein binding to large unilamellar vesicles containing these lipids

• Domain swapping experiments:

  • Create chimeric proteins with domains swapped between different EXO70 paralogs

  • Express in exo70a1 background and use antibodies to track localization

  • This can identify which domains are necessary for membrane recruitment versus protein-protein interactions

• Temporal analysis of assembly:

  • Use rapid fixation methods after cellular stimulation

  • Perform sequential immunofluorescence with antibodies against different exocyst components

  • This can reveal the order of recruitment to specific cellular locations

How can researchers differentiate between specific and non-specific signals when using EXO70A1 antibodies?

To differentiate between specific and non-specific signals:

• Essential controls:

  • Genetic negative control: Use exo70a1 knockout/knockdown tissues (note that some truncated proteins may still be detected depending on the mutant allele and antibody epitope)

  • Technical negative control: Omit primary antibody or use pre-immune serum

  • Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

• Signal validation approaches:

  • Compare signals from multiple independently generated antibodies targeting different epitopes

  • Correlate antibody signals with fluorescently tagged EXO70A1 in transgenic lines

  • Verify predicted molecular weight (approximately 76 kDa for Arabidopsis EXO70A1)

• Challenging cases:

  • In some cases, antibodies may detect signals in muscles even in exo70 mutants, indicating non-specific background that persists despite knockdown

  • When possible, use alternative approaches like in situ hybridization to corroborate protein localization patterns

• Analysis strategies:

  • Normalize EXO70A1 protein Western blot band intensity against internal controls like actin

  • Use quantitative image analysis with appropriate background subtraction

How should researchers interpret discrepancies between antibody-based detection and fluorescent protein fusion localization patterns?

When faced with discrepancies between antibody staining and fluorescent protein localization:

• Common causes of discrepancy:

  • Epitope masking: The antibody epitope may be obscured in certain cellular contexts

  • Fixation artifacts: Some localization patterns may be sensitive to fixation methods

  • Overexpression effects: Fluorescent fusion proteins may mislocalize if overexpressed

  • Tag interference: The fluorescent tag may interfere with protein interactions or localization

• Resolution strategies:

  • Test multiple fixation and permeabilization protocols

  • Compare N- and C-terminal fluorescent protein fusions

  • Use different promoters to achieve more native expression levels

  • Perform antibody staining on tissues expressing the fluorescent fusion to directly compare patterns

• Validation approaches:

  • Functional complementation: Verify that fluorescent fusion proteins rescue mutant phenotypes

  • Biochemical fractionation: Compare distribution of native protein (by antibody) and fusion protein

  • Super-resolution microscopy: Higher resolution may resolve apparent discrepancies in localization patterns

• Interpretation framework:

  • Consider that different detection methods may reveal different subpopulations of the protein

  • Temporal dynamics may explain some discrepancies (antibody staining provides a snapshot while live imaging shows dynamics)

  • Both methods may be correct but revealing different aspects of protein biology

What factors should be considered when analyzing EXO70A1 expression levels in different experimental conditions?

When analyzing EXO70A1 expression levels:

• Tissue-specific considerations:

  • EXO70 genes show highly specific expression patterns; none of the 22 EXO70 genes examined were constitutively expressed

  • EXO70A1 is primarily expressed in exocytosis-active cells, including root tips, developing xylem, and expanding cells

  • Always consider the appropriate tissue controls based on known expression patterns

• Developmental timing:

  • Expression can vary dramatically during development

  • For instance, EXO70B2 protein significantly increases at 4 hours post-inoculation with pathogens, while EXO70A1 slightly decreases

  • Use time-course experiments to capture dynamic changes

• Environmental factors:

  • Stress responses can alter EXO70 paralog expression patterns

  • When comparing treatments, ensure consistent growth and sampling conditions

  • Consider circadian effects on expression levels

• Quantification methods:

  • For Western blots, use appropriate normalization controls (e.g., actin, GAPDH)

  • For qPCR, validate reference genes for stability under your experimental conditions

  • When possible, use multiple detection methods (protein and mRNA levels)

What are the key considerations for using EXO70A1 antibodies in cross-species studies?

For cross-species studies with EXO70A1 antibodies:

• Epitope conservation analysis:

  • Perform sequence alignment of the antibody epitope region across target species

  • The QR motif near the C-terminus is highly conserved and may be a good target for cross-species antibodies

  • Structural conservation may be more important than sequence identity; the EXO70A1 structure revealed high structural similarity with mouse EXO70 despite low sequence identity (32% in middle C-terminal domains)

• Validation requirements:

  • Always validate antibodies in each new species with appropriate positive and negative controls

  • Western blot should show bands of the expected molecular weight

  • Consider producing species-specific antibodies for critical experiments

• Cross-reactivity considerations:

  • Be aware that antibodies may cross-react with other EXO70 paralogs in the same species

  • Sequence identities between members range from 15.8% to 76.5%

  • Use exo70a1 mutants as negative controls where available

• Alternative approaches:

  • For new species, consider creating epitope-tagged versions of EXO70A1

  • Use conserved antibodies against other exocyst subunits as complementary markers

  • When possible, combine antibody approaches with genomic and transcriptomic data

How can EXO70A1 antibodies be used to investigate the role of the exocyst in plant-specific processes like cell wall formation?

To investigate exocyst roles in cell wall formation:

• Co-localization with cell wall synthesis machinery:

  • Use dual immunolabeling with anti-EXO70A1 and antibodies against cellulose synthase complex (CSC) components

  • Recent research showed EXO70A1 is crucial for tethering CSCs to the plasma membrane; exo70a1 mutants exhibited decreased crystalline cellulose content and reduced density of functional CSCs in the plasma membrane

• Temporal analysis during cell wall regeneration:

  • Use systems like protoplast cell wall regeneration

  • Track EXO70A1 localization during different phases of wall formation

  • Correlate with deposition of different cell wall components

• Stress response analysis:

  • Examine EXO70A1 localization during cell wall stress (e.g., isoxaben treatment)

  • Analyze how exocyst components redistribute during adaptive responses

• Trafficking analysis:

  • Use vesicle purification followed by immunoblotting to identify EXO70A1-positive vesicle populations

  • Compare vesicle cargoes between wild-type and exo70a1 mutants to identify specific trafficking defects

What methodological advances might enhance the specificity and sensitivity of EXO70A1 detection in complex plant tissues?

Future methodological advances may include:

How might EXO70A1 antibodies contribute to understanding evolutionary divergence of exocytosis mechanisms across kingdoms?

EXO70A1 antibodies can contribute to evolutionary studies by:

• Comparative localization studies:

  • Apply validated antibodies across diverse plant lineages

  • Compare with mammalian and fungal systems using homologous antibodies

  • This could reveal conserved and divergent aspects of exocyst localization and function

• Functional conservation analysis:

  • The high structural similarity between plant and mammalian EXO70 (despite only 32% sequence identity) suggests functional conservation

  • Antibody studies can help determine if this conservation extends to interaction partners and regulatory mechanisms

• Subcomplex composition comparison:

  • Immunoprecipitate EXO70A1 from diverse species

  • Analyze associated proteins to determine if exocyst subcomplexes are conserved

  • This could reveal evolutionary pressure points in exocyst complex assembly

• Paralog specialization mapping:

  • In plants with expanded EXO70 families, use paralog-specific antibodies to map functional diversification

  • This approach revealed high functional specialization, with EXO70A1 substitutable only by its closest paralog EXO70A2

What strategies can be developed to study post-translational modifications of EXO70A1 using modified antibodies?

Strategies for studying EXO70A1 post-translational modifications include:

• Modification-specific antibodies:

  • Develop antibodies specific to phosphorylated, ubiquitinated, or otherwise modified EXO70A1

  • Use phospho-proteomic data to identify key modification sites

  • These could reveal how modifications regulate EXO70A1 function or localization

• Sequential immunoprecipitation:

  • First immunoprecipitate with general anti-EXO70A1 antibodies

  • Then probe with modification-specific antibodies (anti-phospho, anti-ubiquitin)

  • Alternatively, re-immunoprecipitate with modification-specific antibodies

• Mass spectrometry validation:

  • Immunoprecipitate EXO70A1 from tissues under different conditions

  • Analyze by mass spectrometry to identify condition-specific modifications

  • Use this data to guide development of modification-specific antibodies

• Functional correlation:

  • Compare modification patterns in wild-type plants versus mutants with altered exocytosis

  • Correlate modifications with specific cellular processes or stress responses

  • This could reveal regulatory mechanisms controlling EXO70A1 activity