PBL13 Antibody

What is PBL13 and why would researchers need antibodies against it?

PBL13 is a serine/threonine protein kinase belonging to the RLCK subfamily VII in Arabidopsis thaliana that negatively regulates plant innate immunity to bacterial pathogens. It possesses a unique domain architecture, including a distinctive 15-amino acid repeat motif (K(P/T)RRE(V/T)K(E/D)TSLQNFD) repeated five times in tandem at its C-terminus .

Researchers would need antibodies against PBL13 for several critical applications:

• Detecting and quantifying PBL13 protein levels in plant tissues

• Studying PBL13's subcellular localization

• Investigating protein-protein interactions via co-immunoprecipitation assays

• Analyzing PBL13's post-translational modifications, particularly phosphorylation states

• Validating knockout or knockdown mutants in functional studies

The importance of PBL13 in plant immunity is underscored by the finding that pbl13 T-DNA mutants exhibit enhanced disease resistance to Pseudomonas syringae pv. tomato DC3000, demonstrating PBL13's role as a negative regulator of plant defense responses .

What are the key considerations for validating PBL13 antibody specificity?

When validating PBL13 antibody specificity, researchers should consider:

• Genetic controls: Test antibody reactivity in wild-type plants versus pbl13 knockout lines (pbl13-2 is a complete knockout) . Absence of signal in knockout lines confirms specificity.

• Protein size verification: Wild-type PBL13 has a predicted molecular weight of approximately 56 kDa but typically migrates at approximately 70 kDa when phosphorylated . This mobility shift is a key characteristic to confirm when validating antibodies.

• Phosphatase treatment: Treatment of samples with phosphatases should eliminate the mobility shift observed with PBL13, providing an additional validation method .

• Recombinant protein controls: Test reactivity against purified recombinant PBL13 and other related RLCKs (particularly RIPK, which shares 85% identity in the kinase domain) .

• Cross-reactivity assessment: Evaluate potential cross-reactivity with closely related RLCKs, especially PBL12 and RIPK, which share significant sequence homology .

How can researchers distinguish between phosphorylated and non-phosphorylated forms of PBL13?

Distinguishing between phosphorylated and non-phosphorylated forms of PBL13 requires specialized techniques:

Table 1: Methods for Detecting PBL13 Phosphorylation States

Based on published research, recombinant PBL13 exhibits a significant mobility shift during SDS-PAGE (running at ~70 kDa instead of its predicted 56 kDa) due to autophosphorylation . This shift is eliminated when samples are treated with calf intestinal phosphatase, confirming that phosphorylation causes the mobility shift . Therefore, the simplest method to distinguish phosphorylation states is to compare migration patterns with and without phosphatase treatment.

For researchers generating phospho-specific antibodies, targeting the unique C-terminal repeat motif may be particularly useful, as this region contains multiple phosphorylation sites .

What approaches enable detection of PBL13-RBOHD interactions using antibodies?

PBL13 has been shown to associate with the NADPH oxidase RBOHD, which is critical for reactive oxygen species (ROS) production during immune responses . To study this interaction:

• Co-immunoprecipitation (Co-IP):

  • Use anti-PBL13 antibodies to pull down PBL13 and detect RBOHD in the immunoprecipitate

  • Alternatively, use anti-RBOHD antibodies to pull down RBOHD and detect PBL13

  • Both approaches have been successfully employed in previous studies

• Proximity ligation assay (PLA):

  • Apply primary antibodies against both PBL13 and RBOHD

  • Use oligonucleotide-conjugated secondary antibodies that generate a fluorescent signal when in close proximity

  • This technique can visualize protein interactions in situ

• Bimolecular fluorescence complementation (BiFC):

  • Express fusion proteins of PBL13 and RBOHD with complementary fragments of a fluorescent protein

  • Reconstitution of fluorescence indicates interaction

  • Previous studies have demonstrated that PBL13 primarily interacts with RBOHD's C-terminus

• Split-luciferase complementation assay:

  • Similar to BiFC but uses luciferase fragments

  • This method has been successfully used to show that flagellin treatment disrupts the PBL13-RBOHD association

When designing experiments to study PBL13-RBOHD interactions, researchers should consider that PBL13 preferentially interacts with RBOHD's C-terminus (amino acids 740-921) rather than its N-terminus .

How should researchers optimize western blotting protocols for detecting PBL13?

Given PBL13's unique properties, researchers should consider these optimization strategies for western blotting:

• Sample preparation:

  • Include phosphatase inhibitors in extraction buffers to preserve phosphorylation states

  • For comparative studies, prepare parallel samples with and without phosphatase treatment to distinguish phosphorylation-dependent changes

• Gel percentage selection:

  • Use 8-10% acrylamide gels to achieve optimal separation around the 56-70 kDa range

  • Consider using Phos-tag™ acrylamide for enhanced separation of phosphorylated species

• Transfer conditions:

  • Optimize transfer time for larger proteins (60-70 kDa)

  • Use methanol-free transfer buffers for improved transfer of phosphorylated proteins

• Blocking solutions:

  • Test both BSA and milk-based blocking solutions (phospho-specific antibodies often perform better with BSA)

  • Consider using specialized blocking reagents for phospho-proteins if working with phospho-specific antibodies

• Controls to include:

  • Recombinant PBL13 (wild-type and kinase-dead K111A variant)

  • Samples from pbl13 knockout plants

  • Phosphatase-treated samples to demonstrate mobility shifts

• Detection strategies:

  • For quantitative analysis, consider fluorescent secondary antibodies rather than chemiluminescence

  • For studying phosphorylation dynamics, reprobe membranes with phospho-specific and total PBL13 antibodies

What controls are essential when studying PBL13-mediated phosphorylation of RBOHD?

When investigating PBL13-mediated phosphorylation of RBOHD, researchers should include these essential controls:

• Genetic controls:

  • Wild-type plants

  • pbl13 knockout mutants (pbl13-2)

  • pbl13 complementation lines expressing PBL13-3xFLAG

  • pbl13 complementation lines expressing kinase-dead PBL13 K111A-3xFLAG

• Phosphorylation site mutants:

  • RBOHD with alanine substitutions at PBL13 phosphorylation sites (S780A, S862A, S907A, T910A, T911A, T912A)

  • RBOHD with phosphomimetic mutations (e.g., S862D, T912D)

• Kinase activity controls:

  • Recombinant wild-type PBL13

  • Kinase-dead PBL13 K111A variant

  • Phosphatase-treated samples

• Phospho-specific antibody controls:

  • Antibodies recognizing phosphorylated RBOHD residues (e.g., α-pS862)

  • Validation using phosphatase-treated samples

  • Validation using RBOHD phosphorylation site mutants (e.g., S862A)

• Functional assays:

  • ROS production measurements to correlate phosphorylation status with RBOHD activity

  • Bacterial infection assays to assess impact on immune responses

Published research has demonstrated that PBL13 phosphorylates RBOHD at multiple sites, with S862 and T912 affecting RBOHD activity and stability, respectively .

Why might researchers observe variable detection of PBL13 in experimental conditions?

Variable detection of PBL13 can occur for several reasons:

• Phosphorylation-dependent stability:

  • Unphosphorylated PBL13 appears to be less stable than phosphorylated forms

  • Kinase-dead PBL13 K111A shows reduced accumulation in E. coli

• Pathogen/PAMP-induced changes:

  • PBL13 expression is induced during treatment with Pto DC3000 (AvrRpm1), Harpin Z, NECROSIS-INDUCING PHYTOPHTHORA PROTEIN1, and P. syringae pv. phaseolicola

  • Flagellin treatment disrupts the association between PBL13 and RBOHD

• Tissue-specific expression patterns:

  • Expression levels may vary across different plant tissues

  • Regulatory mechanisms may differ between tissues

• Protein degradation:

  • PBL13 may be subject to regulated protein turnover

  • The E3 ubiquitin ligase PIRE interacts with PBL13 and may regulate its stability

• Technical considerations:

  • Extraction methods may affect protein recovery

  • The unique repeat motif in PBL13's C-terminus may affect antibody accessibility

To address variability, researchers should standardize extraction protocols, include appropriate controls, and consider using epitope-tagged PBL13 variants in complementation lines when possible .

How can researchers interpret contradictory results between PBL13 antibody detection and functional assays?

When faced with contradictory results between antibody detection and functional assays, consider these analytical approaches:

• Assess protein functionality vs. abundance:

  • PBL13's function depends on its kinase activity, not just presence

  • Kinase-dead PBL13 K111A is unable to complement pbl13-2 disease phenotypes, despite protein expression

• Evaluate post-translational modifications:

  • Phosphorylation status affects PBL13's migration pattern and potentially its activity

  • Use phosphatase treatments and Phos-tag gels to distinguish different phosphoforms

• Consider protein-protein interactions:

  • PBL13's association with RBOHD is dynamically regulated by pathogen perception

  • The interaction with PIRE (E3 ubiquitin ligase) may affect PBL13 and RBOHD stability

• Analyze subcellular localization:

  • PBL13's function may depend on proper localization

  • Use cell fractionation or microscopy with antibodies to verify localization

• Examine downstream effects:

  • Measure ROS production, MAPK activation, and defense gene expression as functional readouts

  • Bacterial infection assays provide definitive evidence of immune function

Table 2: Reconciling Contradictory Results in PBL13 Research

The most definitive approach is to combine biochemical analyses with genetic complementation studies, as demonstrated in previous research where wild-type PBL13-3xFLAG, but not kinase-dead PBL13 K111A-3xFLAG, restored wild-type bacterial growth in pbl13-2 mutants .

How can researchers use PBL13 antibodies to investigate the PIRE-PBL13-RBOHD regulatory network?

The discovery of PIRE (PBL13 Interacting RING domain E3 ligase) has revealed a complex regulatory network controlling RBOHD stability and activity . Researchers can use PBL13 antibodies to investigate this network through:

• Triple co-immunoprecipitation studies:

  • Immunoprecipitate PBL13 and probe for both PIRE and RBOHD

  • Determine if these proteins form a tripartite complex or compete for binding

• Sequential immunoprecipitation:

  • First immunoprecipitate with anti-PBL13 antibodies

  • Elute complexes and perform a second immunoprecipitation with anti-PIRE antibodies

  • Analyze if RBOHD is present in both immunoprecipitates

• Proximity-dependent labeling:

  • Fuse PBL13 to biotin ligase (BioID) or APEX2

  • Identify proteins in proximity to PBL13 after activation

  • Compare results between wild-type and treated conditions

• Quantitative co-immunoprecipitation:

  • Perform co-IPs under different conditions (untreated, flagellin-treated, etc.)

  • Quantify relative amounts of PIRE and RBOHD associated with PBL13

  • Correlate changes with functional outcomes (ROS production, disease resistance)

• Ubiquitination assays:

  • Immunoprecipitate RBOHD and analyze ubiquitination status

  • Compare between wild-type, pbl13 mutants, and pire mutants

  • Investigate how PBL13-mediated phosphorylation affects PIRE-dependent ubiquitination

Previous research has demonstrated that PIRE and PBL13 mutants display higher RBOHD protein accumulation, increased ROS production, and enhanced resistance to bacterial infection , suggesting that this regulatory network is critical for fine-tuning immune responses.

What are the methodological considerations for developing phospho-specific antibodies against PBL13 phosphorylation sites?

Developing phospho-specific antibodies for PBL13 requires careful consideration of several factors:

• Phosphorylation site selection:

  • Focus on sites with confirmed biological significance

  • PBL13 undergoes significant autophosphorylation, particularly at its unique C-terminal repeat region

  • Consider sites that undergo dynamic changes during immune responses

• Peptide design considerations:

  • Include 10-15 amino acids surrounding the phosphorylation site

  • Ensure peptide includes unique sequences to avoid cross-reactivity

  • For the repeat motif (K(P/T)RRE(V/T)K(E/D)TSLQNFD), carefully design peptides that can distinguish between individual repeats

• Validation methodology:

  • Test against recombinant PBL13 with and without phosphatase treatment

  • Validate using kinase-dead PBL13 K111A as a negative control

  • Confirm specificity using phosphorylation site mutants (e.g., S→A mutations)

• Application-specific considerations:

  • For western blotting, optimize blocking conditions (BSA often works better than milk for phospho-antibodies)

  • For immunoprecipitation, consider crosslinking antibodies to beads to avoid heavy chain interference

  • For immunofluorescence, optimize fixation methods to preserve phospho-epitopes

• Controls for experimental use:

  • Include lambda phosphatase-treated samples as negative controls

  • Use PBL13 overexpression lines as positive controls

  • Include pbl13 knockout tissues as specificity controls

Table 3: Potential PBL13 Phosphorylation Sites for Antibody Development

When using phospho-specific antibodies, researchers should always run parallel blots with antibodies against total PBL13 to normalize for protein abundance changes.