D6PK Antibody

What is D6PK and why are antibodies against it valuable for plant research?

D6PK is a plasma membrane-associated protein kinase that activates PIN-FORMED (PIN) auxin transporters through direct phosphorylation. D6PK moves rapidly to and from the plasma membrane and contains a middle domain that is required and sufficient for its association and polarity maintenance at the plasma membrane . Antibodies against D6PK are valuable research tools because they allow detection of the protein's subcellular localization, abundance, and phosphorylation status. These antibodies enable researchers to study the mechanisms by which D6PK regulates auxin transport, a fundamental process in plant growth and development . Unlike other PIN-phosphorylating kinases such as PINOID (PID), D6PK doesn't alter PIN polarity but rather activates PIN-mediated auxin efflux directly, making it an important subject for investigating distinct regulatory mechanisms in auxin transport .

How do I validate the specificity of a D6PK antibody in plant tissues?

To validate a D6PK antibody's specificity in plant tissues, implement the following methodological approach:

• Use d6pk knockout mutants as negative controls - antibody signals should be absent or significantly reduced in these tissues compared to wild-type .

• Perform Western blot analysis comparing wild-type and d6pk mutant samples to confirm the antibody detects a protein of the expected molecular weight (~72-75 kDa) only in wild-type samples.

• Conduct immunolocalization experiments in tissues where D6PK is known to be expressed (such as hypocotyls or root cells) to verify that the staining pattern matches the expected basal plasma membrane localization .

• Include peptide competition assays where pre-incubation of the antibody with the peptide used for immunization should abolish or reduce the signal.

• If using phospho-specific D6PK antibodies, treat samples with phosphatase inhibitors and compare with phosphatase-treated samples to confirm phosphorylation-dependent detection.

What controls should I include when using D6PK antibodies for immunolocalization?

When performing immunolocalization with D6PK antibodies, include these essential controls:

• Genetic controls: Include d6pk single, double or triple mutants (d6pk012) as negative controls . The observed signal should be absent or significantly reduced in these genetic backgrounds.

• Treatment controls: Use Brefeldin A (BFA) treatment, which causes D6PK to rapidly internalize from the plasma membrane into BFA bodies. This serves as a positive control for antibody functionality since the signal should relocate from the plasma membrane to intracellular compartments within minutes of treatment .

• Specificity controls: Include secondary antibody-only controls to rule out non-specific binding of the secondary antibody.

• Cross-reactivity controls: If studying specific D6PK family members (D6PK, D6PKL1, D6PKL2, D6PKL3), use the respective single mutants to confirm specificity of the antibody for the intended D6PK family member .

• Phosphorylation state controls: When using phospho-specific antibodies, include samples treated with protein kinase inhibitors or phosphatase treatments to verify phosphorylation-dependent detection .

How should I optimize fixation and extraction conditions for D6PK immunostaining?

Optimizing fixation and extraction conditions for D6PK immunostaining requires careful consideration of the protein's membrane association properties:

• Fixation: Use 4% paraformaldehyde in PBS or MTSB (microtubule stabilizing buffer) for 30-60 minutes. D6PK associates with the plasma membrane through both electrostatic interactions with phospholipids and S-acylation of cysteine residues in its CXX(X)P motifs , so proper fixation is crucial to maintain these associations.

• Membrane preservation: Add 0.1-0.25% Triton X-100 for controlled permeabilization. Excessive detergent may disrupt D6PK's membrane associations, particularly since the protein cycles between the plasma membrane and intracellular compartments .

• Antigen retrieval: Consider mild heat treatment or enzymatic digestion of cell walls if working with intact plant tissues, as this may improve antibody accessibility.

• Blocking: Use 3-5% BSA or normal serum from the species in which the secondary antibody was raised to reduce background.

• Incubation conditions: Optimize primary antibody concentration and incubation time (typically 1:100-1:500, overnight at 4°C) to achieve the best signal-to-noise ratio.

• Buffer considerations: Since D6PK interacts with phosphoinositides through a polybasic motif , avoid buffers that may interfere with these interactions during the initial fixation steps.

How can I distinguish between the phosphorylation activities of D6PK versus other PIN-phosphorylating kinases using antibodies?

Distinguishing between the phosphorylation activities of D6PK and other PIN-phosphorylating kinases (such as PID/WAGs) requires a strategic approach utilizing phosphosite-specific antibodies:

• Phosphosite-specific antibodies: Use antibodies that specifically recognize the distinct phosphorylation sites on PIN proteins. D6PK preferentially phosphorylates PIN1 at serines S1, S4, and S5, while PID/WAGs show preference for S1, S2, and S3 . A phosphosite-specific antibody for PIN1 S4 can effectively detect D6PK-dependent phosphorylation, as this site is primarily targeted by D6PK but not efficiently by PID/WAGs .

• Differential BFA sensitivity: D6PK is highly sensitive to BFA treatment and rapidly internalizes from the plasma membrane, while PID/WAGs are relatively BFA-insensitive . Therefore, PIN phosphorylation at the basal membrane that decreases rapidly after BFA treatment is indicative of D6PK activity, while phosphorylation that persists after BFA treatment, particularly at the apical membrane, suggests PID/WAG activity .

• Genetic background analysis: Compare phosphorylation patterns in wild-type, d6pk multiple mutants, and pid/wag mutant backgrounds. Phosphorylation dependent on D6PK will be reduced or absent in d6pk mutants but maintained in pid/wag mutants, and vice versa .

• Subcellular localization: Immunolocalization experiments reveal that D6PK-dependent phosphorylation predominantly occurs at the basal plasma membrane, whereas PID/WAG-dependent phosphorylation is observed at both apical and basal membranes . This spatial distribution can help distinguish between the activities of these kinases.

• Functional readouts: Combine phosphorylation detection with functional assays, as D6PK primarily activates PIN-mediated auxin efflux without changing PIN polarity, while PID/WAGs induce a basal-to-apical polarity shift .

What approaches can resolve contradictory immunolocalization data regarding D6PK membrane association patterns?

When faced with contradictory immunolocalization data regarding D6PK membrane association patterns, implement these methodological approaches:

• Multiple fixation protocols: Compare different fixation methods (chemical cross-linking versus rapid freezing/freeze substitution) as membrane-associated proteins like D6PK can be sensitive to fixation artifacts. The S-acylation of D6PK's CXX(X)P motifs is critical for membrane association , and certain fixation protocols may disrupt these modifications.

• Live-cell imaging validation: Compare immunolocalization results with live-cell imaging of fluorescently tagged D6PK (YFP-D6PK) expressed under native promoters (D6PKp:YFP:D6PK) . This approach avoids fixation artifacts completely and allows dynamic tracking of D6PK localization.

• Multiple antibody approaches: Use both N-terminal and C-terminal targeted antibodies against D6PK, as well as antibodies recognizing specific post-translational modifications. Different epitopes may be differentially accessible depending on D6PK's conformational state or interaction partners.

• Membrane fractionation: Complement immunolocalization with biochemical fractionation techniques to quantitatively assess D6PK distribution between membrane and cytosolic fractions under different conditions.

• Pharmacological interventions: Use treatments that affect specific aspects of D6PK localization:

  • 2-Bromopalmitate (2-BP) to inhibit S-acylation

  • BFA to block vesicle trafficking

  • Phosphatase inhibitors to stabilize phosphorylated forms of D6PK
    These interventions can help identify the specific mechanisms contributing to observed localization patterns.

• Genetic manipulation: Compare D6PK localization in mutants affecting potential regulatory mechanisms, such as:

  • Mutations in CXX(X)P motif cysteines (C1S through C5S)

  • Mutations in S310/S311 phosphorylation sites (SSAA, SSDD)

  • Mutations in the polybasic motif for phosphoinositide interactions

How should I design experiments to investigate the relationship between D6PK S-acylation and phosphorylation using specific antibodies?

To investigate the relationship between D6PK S-acylation and phosphorylation using specific antibodies, design your experiments as follows:

• Generate/obtain specific antibodies:

  • Anti-D6PK antibodies for total protein detection

  • Phospho-specific antibodies targeting S310/S311, which are important for D6PK trafficking

  • Antibodies that can distinguish between S-acylated and non-acylated forms of D6PK (challenging but potentially achievable using specialized protocols)

• Sequential analysis approach:

  • Use the acyl-biotinyl exchange (ABE) or acyl-resin-assisted capture (acyl-RAC) methods to isolate S-acylated proteins

  • Follow with immunoblotting using phospho-specific antibodies to determine the correlation between S-acylation and phosphorylation states

  • Compare wild-type D6PK with cysteine mutants (C1S-C5S) that show reduced S-acylation

• Pharmacological interventions:

  • Treat samples with 2-Bromopalmitate (2-BP) to inhibit S-acylation and examine effects on phosphorylation levels

  • Use kinase inhibitors or phosphatase treatments to examine effects on S-acylation

  • Apply BFA treatment to examine trafficking dynamics in relation to these modifications

• Mobility shift analysis:

  • Perform high-resolution SDS-PAGE and immunoblotting with anti-D6PK antibodies

  • Compare migration patterns of wild-type versus D6PK variants with mutations in CXX(X)P motifs (C1S-C5S) and phosphorylation sites (SSAA, SSDD)

  • Include treatments with phosphatase or deacylation reagents to confirm band identities

• Microscopy correlation studies:

  • Perform dual immunofluorescence experiments using antibodies against D6PK and membrane/trafficking markers

  • Compare localization patterns of wild-type versus mutant forms (C1S-C5S, SSAA, SSDD)

  • Quantify co-localization coefficients to establish relationships between modifications and localization

• Mass spectrometry validation:

  • Immunoprecipitate D6PK from various conditions and analyze by mass spectrometry

  • Identify peptides with S-acylation and phosphorylation modifications

  • Determine whether these modifications occur simultaneously or are mutually exclusive

What are the critical parameters for designing an immunoassay to detect D6PK-dependent PIN phosphorylation in plant samples?

Designing an effective immunoassay to detect D6PK-dependent PIN phosphorylation requires attention to several critical parameters:

• Phosphosite-specific antibody selection:

  • Use antibodies that specifically recognize PIN phosphosites preferentially targeted by D6PK (S4 and S5) rather than those predominantly phosphorylated by PID/WAGs

  • Validate antibody specificity using phospho-null mutants (e.g., PIN1 S4A) as negative controls

  • Consider generating antibodies against multiply phosphorylated epitopes if D6PK acts on adjacent sites simultaneously

• Sample preparation optimization:

  • Rapidly harvest and flash-freeze tissues to preserve phosphorylation status

  • Include phosphatase inhibitors (e.g., NaF, Na₃VO₄) in all extraction buffers

  • Use mild detergents that solubilize membrane proteins without disrupting protein complexes

  • Consider subcellular fractionation to enrich for plasma membrane fractions where D6PK-PIN interactions occur

• Assay controls:

  • Include d6pk multiple mutant samples as negative controls

  • Use samples treated with lambda phosphatase to confirm phosphorylation-dependent signals

  • Include BFA-treated samples to distinguish D6PK-dependent phosphorylation (rapidly lost after BFA) from PID/WAG-dependent phosphorylation (BFA-resistant)

  • Compare wild-type with PIN phosphosite mutants (S4A, S5A) to confirm signal specificity

• Quantification approach:

  • Use calibration curves with known amounts of phosphorylated peptides

  • Normalize phospho-PIN signals to total PIN protein levels

  • Consider multiplexed detection of multiple phosphosites simultaneously

  • Implement image analysis software for consistent quantification of immunofluorescence signals

• Physiological relevance:

  • Correlate PIN phosphorylation levels with auxin transport activity measurements

  • Compare phosphorylation patterns across different tissues and developmental stages

  • Assess phosphorylation dynamics in response to environmental stimuli that affect auxin transport

How can I use D6PK antibodies to investigate the mechanisms of phosphoinositide-dependent membrane domain formation?

To investigate phosphoinositide-dependent membrane domain formation using D6PK antibodies, implement the following experimental strategies:

• Co-localization studies:

  • Use dual immunolabeling with D6PK antibodies and antibodies or probes against specific phosphoinositides

  • Focus particularly on PtdIns(4)P, which has been implicated in D6PK membrane association through its polybasic motif

  • Quantify co-localization at the basal plasma membrane and in developing pollen where D6PKL3 forms distinct membrane domains

• Domain perturbation analysis:

  • Generate plants expressing D6PK variants with mutations in the polybasic K/R-rich motif that mediates electrostatic interactions with phosphoinositides

  • Compare membrane domain formation using antibodies against wild-type and mutant D6PK

  • Correlate domain formation with functional outputs such as PIN phosphorylation and auxin transport efficiency

• Phosphoinositide manipulation:

  • Treat plants with phosphoinositide metabolism inhibitors (e.g., wortmannin for PI3K inhibition)

  • Apply exogenous phosphoinositides in membrane permeabilized cells

  • Express phosphoinositide-specific phosphatases to deplete specific phosphoinositide pools

  • After each manipulation, use D6PK antibodies to assess effects on domain formation and maintenance

• Super-resolution microscopy:

  • Apply techniques such as STORM, PALM, or STED microscopy with D6PK antibodies

  • Measure the size, distribution, and dynamics of D6PK-containing membrane domains

  • Compare wild-type patterns with those in plants with altered phosphoinositide composition

• Biochemical domain isolation:

  • Use detergent-resistant membrane fractionation to isolate potential lipid rafts or microdomains

  • Immunoblot fractions with D6PK antibodies to determine domain association

  • Analyze lipid composition of D6PK-containing fractions to identify critical phosphoinositides

• Protein interaction network analysis:

  • Use D6PK antibodies for co-immunoprecipitation studies to identify interaction partners

  • Focus on proteins involved in phosphoinositide metabolism or membrane domain organization

  • Correlate with known domain formation factors like INAPERTURATE POLLEN1, which fails to aggregate at aperture sites in d6pkl3 mutants

What are the optimal conditions for using D6PK antibodies in immunoprecipitation experiments?

For optimal immunoprecipitation of D6PK, implement these methodological considerations:

• Buffer composition:

  • Use mild lysis buffers (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40 or 0.5% Triton X-100)

  • Include protease inhibitors (PMSF, leupeptin, aprotinin, pepstatin A)

  • Add phosphatase inhibitors (NaF, Na₃VO₄) to preserve phosphorylation status

  • Consider including 10% glycerol to stabilize protein complexes

  • For membrane-associated D6PK, include 1-2 mM MgCl₂ to stabilize membrane interactions

• Sample preparation:

  • Harvest tissues rapidly and flash-freeze in liquid nitrogen

  • Grind thoroughly in liquid nitrogen before adding lysis buffer

  • Allow limited extraction time (15-30 minutes) at 4°C with gentle agitation

  • Clear lysates by centrifugation at 14,000 × g for 15 minutes at 4°C

• Antibody selection and coupling:

  • Use antibodies raised against N-terminal epitopes of D6PK, as the C-terminus may be involved in protein interactions

  • Pre-couple antibodies to protein A/G beads or magnetic beads for more efficient pulldowns

  • Consider using 1-5 μg antibody per mg of total protein

  • Include IgG from the same species as a negative control

• IP conditions:

  • Incubate lysates with antibody-coupled beads for 2-4 hours or overnight at 4°C with gentle rotation

  • Wash beads 4-5 times with wash buffer (lysis buffer with reduced detergent)

  • Include a final wash with detergent-free buffer to remove residual detergent

  • Elute proteins with either low pH (glycine buffer, pH 2.5) followed by immediate neutralization, or with SDS sample buffer

• Special considerations for D6PK:

  • Since D6PK associates with membranes through S-acylation of cysteines in its CXX(X)P motifs , avoid reducing agents like DTT or β-mercaptoethanol in early extraction steps

  • Consider crosslinking approaches to capture transient interactions with trafficking components

  • For studying D6PK-PIN interactions, co-immunoprecipitation may require membrane-permeable crosslinkers applied before cell lysis

How can I use D6PK antibodies to quantitatively assess changes in PIN phosphorylation under different experimental conditions?

To quantitatively assess changes in PIN phosphorylation using D6PK antibodies, implement this methodological framework:

• Phosphospecific ELISA development:

  • Coat plates with anti-PIN antibodies to capture total PIN proteins

  • Detect with phosphosite-specific antibodies that recognize D6PK-dependent phosphorylation sites (S4, S5)

  • Develop standard curves using synthetic phosphopeptides

  • Normalize phospho-signal to total PIN content using a dual-detection approach

• Quantitative immunoblotting:

  • Separate proteins by SDS-PAGE and transfer to membranes

  • Probe with phosphosite-specific antibodies (e.g., anti-PIN1 S4)

  • Strip and reprobe with total PIN antibodies

  • Use fluorescently-labeled secondary antibodies for precise quantification

  • Calculate phosphorylation ratio (phospho-PIN/total PIN)

  • Include standard curves with known amounts of phosphorylated and non-phosphorylated recombinant PIN

• Immunofluorescence quantification:

  • Perform immunolocalization with phosphosite-specific antibodies

  • Acquire images under identical exposure settings

  • Measure fluorescence intensity along plasma membrane domains

  • Normalize to membrane markers or total PIN signal

  • Compare signal intensity across experimental conditions

• In-cell Western assay:

  • Grow plant cells in multi-well plates

  • Fix and permeabilize cells

  • Incubate with phosphosite-specific and total PIN antibodies labeled with different fluorophores

  • Quantify signals using an infrared imaging system

  • Calculate phosphorylation ratio across multiple wells/conditions

• Phos-tag SDS-PAGE:

  • Incorporate Phos-tag into polyacrylamide gels to retard phosphorylated proteins

  • Separate protein extracts and perform immunoblotting with total D6PK or PIN antibodies

  • Quantify the proportion of protein in phosphorylated versus non-phosphorylated bands

  • Compare band patterns across experimental conditions

• Experimental manipulations to assess:

  • BFA treatment time courses to correlate D6PK plasma membrane depletion with PIN phosphorylation

  • 2-BP treatment to inhibit S-acylation and assess effects on D6PK localization and PIN phosphorylation

  • Expression of phosphomimetic (SSDD) versus phospho-null (SSAA) D6PK variants

  • Auxin treatments to determine if PIN phosphorylation is regulated by hormonal stimuli

What are the key considerations when using phospho-specific antibodies to distinguish between D6PK-mediated versus PINOID-mediated PIN phosphorylation?

When using phospho-specific antibodies to distinguish between D6PK-mediated and PINOID-mediated PIN phosphorylation, consider these critical factors:

• Phosphosite specificity and preference:

  • D6PK preferentially phosphorylates PIN1 at serines S1, S4, and S5, while PID/WAGs show preference for S1, S2, and S3

  • Use antibodies specifically targeting S4 to detect primarily D6PK-dependent phosphorylation

  • Use antibodies targeting S2 to detect primarily PID/WAG-dependent phosphorylation

  • For sites phosphorylated by both kinases (S1, S3), additional approaches are needed for discrimination

• Experimental design for differentiation:

  • Perform parallel experiments in d6pk multiple mutants and pid/wag mutants

  • Use BFA treatment as a discriminating tool - D6PK-dependent phosphorylation is rapidly lost after BFA treatment while PID/WAG-dependent phosphorylation is relatively BFA-resistant

  • Compare phosphorylation patterns in tissues where one kinase is predominantly expressed over the other

• Controls and validation:

  • Include phosphosite mutant versions of PIN proteins (S1A, S2A, S3A, S4A, S5A) to validate antibody specificity

  • Perform in vitro kinase assays with recombinant D6PK and PID kinases to establish clear phosphorylation patterns for reference

  • Use phosphatase treatments to confirm that the detected signals are indeed phosphorylation-dependent

• Localization-based discrimination:

  • Perform high-resolution immunolocalization to capitalize on the different subcellular distribution of these kinases

  • D6PK localizes predominantly to the basal plasma membrane, while PID can be found at both apical and basal domains

  • Phosphorylation at the apical plasma membrane that persists after BFA treatment is likely PID/WAG-dependent

• Functional correlation:

  • Correlate phosphorylation patterns with functional outcomes

  • D6PK primarily activates PIN-mediated auxin efflux without changing PIN polarity

  • PID/WAGs induce a basal-to-apical polarity shift in addition to activating transport

  • These different functional outcomes can help interpret ambiguous phosphorylation data

• Quantitative analysis:

  • Implement ratiometric approaches comparing phosphorylation at different sites

  • Calculate the ratio of S4 phosphorylation (D6PK-preferred) to S2 phosphorylation (PID-preferred)

  • Use these ratios to infer the relative contributions of each kinase to the observed phosphorylation pattern

What are common pitfalls when using D6PK antibodies in immunolocalization experiments and how can they be addressed?

Common pitfalls in D6PK immunolocalization experiments and their solutions include:

• High background signal:

  • Problem: Non-specific binding of antibodies to cell walls or other structures.

  • Solution: Optimize blocking conditions (use 3-5% BSA or normal serum); increase washing steps; pre-absorb antibodies with plant powder from d6pk mutants; use highly purified antibody preparations or consider affinity purification.

• Loss of membrane localization:

  • Problem: D6PK's membrane association depends on both S-acylation of cysteines in CXX(X)P motifs and electrostatic interactions with phosphoinositides , which can be disrupted during fixation.

  • Solution: Use milder fixatives (reduce paraformaldehyde concentration to 2-3%); add divalent cations (1-2 mM Mg²⁺) to stabilize phosphoinositide interactions; reduce detergent concentration during permeabilization; consider alternative fixation methods like freeze substitution.

• Variable signal intensity across experiments:

  • Problem: D6PK trafficking is highly dynamic and sensitive to environmental conditions .

  • Solution: Standardize growth conditions and sample harvesting times; fix tissues rapidly; include internal reference markers; consider quantitative approaches normalizing D6PK signal to membrane markers.

• Fixation-induced artifacts:

  • Problem: Chemical fixation may alter protein localization or accessibility.

  • Solution: Validate immunolocalization results with live-cell imaging of fluorescently tagged D6PK ; compare multiple fixation protocols; include controls with brief BFA treatments to confirm expected D6PK internalization .

• Inadequate detection of phosphorylated forms:

  • Problem: Phosphatases may dephosphorylate D6PK during sample preparation.

  • Solution: Include phosphatase inhibitors (50 mM NaF, 1 mM Na₃VO₄) in all buffers; keep samples cold throughout processing; validate results with phosphomimetic (SSDD) and phospho-null (SSAA) D6PK variants .

• Poor epitope accessibility:

  • Problem: D6PK domains may be masked by protein-protein interactions or conformational states.

  • Solution: Try antibodies targeting different epitopes; consider antigen retrieval techniques; compare native versus denatured immunodetection protocols; use epitope-tagged D6PK versions in parallel.

• Cross-reactivity with other D6PK family members:

  • Problem: Antibodies may recognize related D6PK family proteins (D6PKL1, D6PKL2, D6PKL3) .

  • Solution: Validate antibody specificity using multiple mutant combinations; use peptide competition assays; consider raising antibodies against unique regions of specific family members.

How can I adapt D6PK antibody-based protocols to study protein kinase membrane dynamics in different plant species?

To adapt D6PK antibody-based protocols for studying protein kinase membrane dynamics in different plant species, implement these approaches:

• Antibody validation in new species:

  • Perform sequence alignments to identify conservation of epitopes recognized by existing D6PK antibodies

  • Conduct Western blot analysis to confirm cross-reactivity and specificity

  • Test antibodies on tissues from multiple plant species in parallel with Arabidopsis controls

  • Consider raising new antibodies against conserved epitopes for broader species compatibility

• Species-specific protocol modifications:

  • Adjust tissue fixation conditions based on cell wall composition differences

  • Optimize cell wall digestion protocols using species-appropriate enzyme mixtures

  • Modify permeabilization conditions based on membrane composition differences

  • Adjust antibody concentrations and incubation times for each species

• Leveraging heterologous expression systems:

  • Express fluorescently-tagged D6PK orthologs from different species in Arabidopsis

  • Compare antibody detection with fluorescent protein localization

  • Use this approach to calibrate antibody protocols for new species

  • Consider expressing Arabidopsis D6PK in heterologous systems (e.g., Nicotiana benthamiana) as an intermediate step

• Combining with species-specific markers:

  • Identify membrane domain markers for each plant species

  • Use dual-labeling with these markers to normalize D6PK localization patterns

  • Develop species-specific plasma membrane isolation protocols for biochemical studies

  • Utilize conserved cellular structures as internal landmarks across species

• Addressing technical challenges in different tissues:

  • For woody species: Develop specialized fixation protocols to penetrate lignified tissues

  • For species with high phenolic content: Include polyvinylpyrrolidone (PVP) or polyvinylpolypyrrolidone (PVPP) in extraction buffers

  • For species with complex cell walls: Optimize enzymatic digestion or use mechanical sectioning

  • For highly vacuolated cells: Adjust osmotic conditions during fixation to prevent plasmolysis

• Comparative evolutionary analysis:

  • Study conservation of key regulatory features across species:

    • CXX(X)P motifs for S-acylation

    • Polybasic motifs for phosphoinositide interactions

    • S310/S311 equivalents for phosphorylation-dependent trafficking

  • Correlate structural conservation with functional conservation using combined antibody detection and functional assays

What approaches can minimize non-specific background when using phospho-specific D6PK or PIN antibodies in plant tissues?

To minimize non-specific background when using phospho-specific D6PK or PIN antibodies in plant tissues, implement these methodological strategies:

• Antibody purification enhancements:

  • Perform affinity purification of antibodies against the specific phosphopeptide

  • Include a negative selection step using the non-phosphorylated peptide to remove antibodies that recognize the unmodified epitope

  • Consider using monoclonal antibodies for increased specificity when available

  • Validate purified antibodies using phosphatase-treated samples and phospho-null mutants (e.g., PIN1 S4A)

• Optimized blocking strategies:

  • Use a dual blocking approach with 3-5% BSA followed by 5-10% normal serum from the species in which the secondary antibody was raised

  • Add 0.1-0.5% non-ionic detergent (Triton X-100) to blocking solutions to reduce hydrophobic interactions

  • Include 50-100 mM glycine to block residual aldehyde groups from fixation

  • Consider using plant-derived blocking reagents from unrelated species to reduce plant-specific background

• Pre-absorption techniques:

  • Pre-incubate antibodies with plant tissue extracts from phospho-null mutants

  • Use tissue powder from appropriate knockout mutants (d6pk for D6PK antibodies)

  • Include excess non-phosphorylated peptide during antibody incubation to compete away non-phospho-specific binding

  • Validate the specificity of pre-absorbed antibodies using Western blots

• Signal-to-noise enhancement strategies:

  • Implement tyramide signal amplification (TSA) to amplify specific signals while keeping antibody concentrations low

  • Use fluorophores with narrow emission spectra to reduce bleed-through

  • Apply spectral unmixing during image acquisition to separate specific signal from autofluorescence

  • Consider quantum dots as alternative labels for improved signal stability and reduced background

• Tissue preparation refinements:

  • Minimize autofluorescence by treating sections with sodium borohydride or Sudan Black B

  • Include photobleaching steps before antibody incubation

  • Optimize tissue thickness (thinner sections often yield better signal-to-noise ratios)

  • Consider mechanical sectioning methods (vibratome) rather than chemical clearing for certain tissues

• Controls and validation:

  • Always include phosphatase-treated samples as negative controls

  • Use phosphomimetic (S→D) and phospho-null (S→A) mutants for specificity verification

  • Compare BFA-treated versus untreated samples to validate D6PK-dependent phosphorylation

  • Implement tissue-specific expression controls (using tissue-specific promoters) to validate signal specificity in different cell types

How can I design experiments using D6PK antibodies to investigate the relationship between auxin transport and plant development?

To investigate the relationship between auxin transport and plant development using D6PK antibodies, design experiments following these methodological approaches:

• Developmental expression and localization analysis:

  • Map D6PK expression and subcellular localization across different developmental stages using immunohistochemistry

  • Correlate D6PK expression patterns with auxin-responsive reporter lines (DR5, DII-VENUS)

  • Analyze PIN phosphorylation status (using phosphosite-specific antibodies) in parallel with auxin distribution patterns

  • Focus on developmental processes known to be auxin-dependent, such as embryogenesis, apical hook formation, lateral root development, and vascular patterning

• Perturbation experiments:

  • Compare wild-type plants with d6pk multiple mutants and PIN phosphosite mutants

  • Use inducible expression systems for rapid D6PK activation/inactivation and track subsequent developmental changes

  • Apply auxin transport inhibitors (NPA, TIBA) and examine effects on D6PK localization and PIN phosphorylation

  • Manipulate D6PK membrane association through targeted mutations in CXX(X)P motifs or polybasic domains and assess developmental consequences

• Tissue-specific manipulation:

  • Implement tissue-specific complementation of d6pk mutants

  • Compare D6PK and PIN phosphorylation patterns between tissues with different growth responses

  • Use tissue-specific inhibition of D6PK through artificial microRNAs or CRISPR interference

  • Correlate tissue-specific D6PK activity with local auxin transport rates and developmental outputs

• Environmental response studies:

  • Examine how environmental stimuli (light, gravity, touch) affect D6PK localization and PIN phosphorylation

  • Correlate changes in D6PK membrane association with tropic growth responses

  • Use phosphosite-specific antibodies to track rapid changes in PIN phosphorylation during environmental adaptation

  • Combine with auxin transport measurements to establish functional links

• Quantitative correlation approaches:

  • Develop quantitative immunodetection methods to measure D6PK abundance and PIN phosphorylation levels

  • Correlate these measurements with auxin transport rates measured by radioactive auxin transport assays

  • Implement mathematical modeling to predict developmental outcomes based on D6PK-mediated PIN activation

  • Design genetic experiments to test model predictions

• Multi-scale integration:

  • Link molecular-level observations (D6PK localization, PIN phosphorylation) to cellular-level processes (auxin transport, cell expansion)

  • Connect cellular processes to tissue-level patterning and organ development

  • Develop tissue-specific markers for D6PK activity (e.g., using split-GFP approaches) that can be visualized during development

  • Combine with computational modeling to understand how local D6PK-dependent auxin transport affects global developmental patterning

What experimental design would best investigate the interplay between D6PK S-acylation, membrane association, and kinase activity?

To investigate the interplay between D6PK S-acylation, membrane association, and kinase activity, implement this comprehensive experimental design:

• Structure-function analysis with site-directed mutagenesis:

  • Generate a series of D6PK variants with mutations in:

    • Individual or combined CXX(X)P motif cysteines (C1S through C5S)

    • The polybasic lysine/arginine-rich motif for phosphoinositide binding

    • The S310/S311 phosphorylation sites (SSAA and SSDD variants)

    • The ATP-binding site (kinase-dead version)

  • Express these variants under the native D6PK promoter in d6pk multiple mutant backgrounds

  • Assess each variant for membrane association, polar localization, and biological activity

• Dynamic correlation studies:

  • Use pharmacological interventions to separately manipulate:

    • S-acylation (2-BP treatment)

    • Phosphorylation (kinase inhibitors)

    • Membrane trafficking (BFA treatment)

  • Track the temporal sequence of changes in:

    • D6PK membrane association (using immunolocalization)

    • PIN phosphorylation status (using phosphosite-specific antibodies)

    • Auxin transport activity (using radioactive auxin transport assays)

  • Establish cause-effect relationships between these processes

• In vitro reconstitution approaches:

  • Purify recombinant D6PK variants with different S-acylation states

  • Prepare artificial membrane systems with defined phosphoinositide composition

  • Measure membrane binding affinities and kinase activities

  • Reconstitute D6PK-PIN interactions on membranes to assess how S-acylation affects substrate recognition and phosphorylation

• Live-cell imaging combined with biochemical analysis:

  • Implement fluorescence recovery after photobleaching (FRAP) to measure D6PK membrane dynamics

  • Compare wild-type D6PK with S-acylation mutants (C1S-C5S)

  • Correlate membrane residence time with kinase activity measured in parallel samples

  • Use ratiometric FRET-based sensors to monitor D6PK-PIN interactions in real-time

• Interactor identification and validation:

  • Perform immunoprecipitation with antibodies against wild-type D6PK and S-acylation mutants

  • Identify differential interactors using mass spectrometry

  • Validate key interactions using in vitro binding assays and co-localization studies

  • Assess how S-acylation affects D6PK's interaction network

• Computational integration:

  • Develop mathematical models incorporating:

    • S-acylation-dependent membrane association rates

    • Membrane diffusion coefficients for different D6PK variants

    • Kinase activity as a function of membrane association

    • PIN phosphorylation dynamics

  • Use experimental data to parameterize and validate the model

  • Use the model to predict how perturbations in S-acylation would affect auxin transport and development

• Correlation with functional outputs:

  • Compare auxin distribution patterns (using DR5 reporters) in plants expressing D6PK variants with different S-acylation potential

  • Measure physiological responses (gravitropism, phototropism) as readouts of functional auxin transport

  • Assess developmental phenotypes (hook formation, hypocotyl elongation) that depend on D6PK-mediated auxin transport