CEPR2 Antibody

What is CEPR2 and why is it significant for plant research?

CEPR2 is a plasma membrane-localized leucine-rich repeat receptor-like kinase (LRR-RLK) that plays crucial roles in balancing growth regulation and stress responses in Arabidopsis. It interacts with PYR/PYL abscisic acid (ABA) receptors to promote their phosphorylation and subsequent degradation under unstressed conditions, whereas ABA inhibits this process . CEPR2 is predominantly expressed in stomatal guard cells and functions redundantly with CEPR1 in mediating CEP peptide perception and immune responses . Understanding CEPR2 function provides insights into how plants coordinate growth, stress tolerance, and immunity.

What are the optimal tissue sources for CEPR2 antibody applications?

Based on expression pattern studies using pCEPR2::NLS-3xmVenus reporter lines, CEPR2 is primarily expressed in stomatal guard cells . Therefore, leaf tissues with abundant stomata represent the optimal source for CEPR2 antibody applications. When designing experiments, researchers should consider this tissue-specific expression pattern, as CEPR2 protein levels may be difficult to detect in whole-plant extracts due to dilution effects from non-expressing tissues.

How can I validate the specificity of a CEPR2 antibody?

A multi-pronged validation approach is recommended:

• Test antibody reactivity against protein extracts from:

  • Wild-type plants

  • cepr2 mutant plants (negative control)

  • CEPR2 overexpression lines (positive control)

• Perform peptide competition assays using the antigenic peptide sequence

• Assess cross-reactivity with related proteins, particularly CEPR1, which shares sequence homology with CEPR2

• Confirm the detection of a protein band of approximately 100-120 kDa, corresponding to the predicted molecular weight of CEPR2

• Verify subcellular localization by immunofluorescence microscopy, which should show plasma membrane localization

What protein extraction protocols maximize CEPR2 recovery for immunoblotting?

As a plasma membrane-localized protein with a transmembrane domain (amino acids 622-641), CEPR2 requires specialized extraction conditions:

This extraction buffer has been successfully used in Co-IP experiments with CEPR2-GFP fusion proteins . Include phosphatase inhibitors when studying CEPR2 phosphorylation status or kinase activity.

How can I study CEPR2-mediated phosphorylation of target proteins?

To investigate CEPR2's kinase activity toward target proteins such as PYLs:

• In vitro kinase assays:

  • Express and purify the CEPR2 kinase domain (avoid the transmembrane domain)

  • Incubate with recombinant substrate proteins (e.g., GST-PYL4)

  • Detect phosphorylation using:

    • ³²P-ATP incorporation

    • Phospho-specific antibodies

    • Mass spectrometry analysis

• In vivo approaches:

  • Compare phosphorylation levels of target proteins between:

    • Wild-type plants

    • CEPR2 overexpression lines

    • cepr2 mutants

  • Use phosphatase treatments as controls

  • Monitor site-specific phosphorylation (e.g., PYL4 at Ser54)

• Evaluate ABA effects on phosphorylation, as ABA has been shown to inhibit CEPR2-mediated phosphorylation of PYLs

What approaches can detect interactions between CEPR2 and its binding partners?

Multiple complementary techniques have been successfully employed:

• Co-immunoprecipitation (Co-IP):

  • CEPR2-GFP fusion proteins can be immunoprecipitated using anti-GFP antibodies

  • Interacting proteins detected with specific antibodies (e.g., anti-Myc for Myc-tagged PYL proteins)

• Pull-down assays:

  • Immobilized GST-PYL proteins with purified CEPR2 kinase domain

  • Include ABA in experimental conditions to evaluate its effect on interactions

• Yeast-based approaches:

  • Membrane-based split-ubiquitin system (MbSUS) for membrane protein interactions

• In planta confirmation:

  • Luciferase complementation imaging (LCI) in Nicotiana benthamiana

  • Bimolecular fluorescence complementation (BiFC)

Why might I observe discrepancies between CEPR2 transcript and protein levels?

Research has demonstrated that CEPR2 protein levels do not always correlate with transcript abundance. For instance, no statistically significant differences in PYL4 transcript levels were observed among CEPR2 overexpression lines, wild-type, and cepr2/pxy/pxl2 mutants, despite clear differences in protein levels . This discrepancy may result from:

• Post-translational regulation mechanisms affecting CEPR2 stability

• Differences in protein turnover rates

• Translation efficiency variations

• Protein degradation through the 26S proteasome and vacuolar pathways

To address this issue, always measure both transcript levels (via RT-PCR/qRT-PCR) and protein levels (via immunoblotting) when studying CEPR2 function.

How can I distinguish between CEPR1 and CEPR2 functions in experimental settings?

Distinguishing between these functionally redundant receptors requires:

• Tissue-specific analysis:

  • CEPR1 is expressed primarily in vasculature

  • CEPR2 is predominantly in guard cells

• Genetic approaches:

  • Compare phenotypes of single mutants (cepr1, cepr2) versus double mutants (cepr1 cepr2)

  • The double mutant shows enhanced susceptibility to pathogens and ABA response phenotypes not evident in single mutants

• Biochemical specificity:

  • CEPR2 phosphorylates PYL4 at Ser54, which may differ from CEPR1 specificity

  • Different binding affinities for CEP peptides (CEPR2ECD binds CEP4 with KD of 9.3 μM)

• Domain-specific constructs to isolate function of each receptor

What factors affect CEPR2 antibody detection in immunolocalization experiments?

Several factors can influence the success of immunolocalization with CEPR2 antibodies:

• Fixation method:

  • Over-fixation may mask epitopes

  • Insufficient fixation may compromise structural integrity

• Membrane permeabilization:

  • Critical for antibody access to membrane-embedded epitopes

  • Excessive detergent may disrupt protein localization

• Expression level variations:

  • CEPR2 is specifically expressed in guard cells

  • Signal may be weak in other cell types

• ABA treatment effects:

  • ABA influences CEPR2 interactions with targets like PYLs

  • May affect epitope accessibility or protein conformation

• Phosphorylation status:

  • As a kinase, CEPR2's phosphorylation state may influence antibody recognition

  • Consider phosphatase treatments as controls

How do I interpret changes in CEPR2 protein stability under different conditions?

The stability of CEPR2 and its target proteins can be assessed using protein degradation assays. Research has shown that:

• Cycloheximide (CHX) chase assays:

  • Treatment with 100 μM CHX blocks protein synthesis

  • PYL4 protein levels diminish faster in CEPR2 overexpression lines compared to wild-type

  • Degradation is slower in cepr2/pxy/pxl2 mutants

• Proteasome and vacuolar pathway inhibition:

  • 100 μM MG132 (26S proteasome inhibitor) slows degradation

  • 100 μM E64d (vacuolar hydrolase inhibitor) also slows degradation

  • Combined treatment with both inhibitors more effectively prevents degradation

• Half-life calculations:

  • Quantify protein levels at different time points after CHX treatment

  • Calculate half-life using appropriate regression methods

This experimental approach has revealed that CEPR2 promotes degradation of PYLs through both the 26S proteasome and vacuolar degradation pathways .

How can I determine if observed phenotypes are due to CEPR2's role in ABA signaling versus immunity?

CEPR2 functions in both abscisic acid signaling and immune responses, making it challenging to attribute phenotypes to specific pathways. A systematic approach includes:

• Pathway-specific markers:

  • ABA pathway: expression of ABI4, ABI5, RAB18

  • Immunity: bacterial growth (PtoCOR-), defense gene expression

• Genetic background considerations:

  • Single versus double mutants (cepr1 cepr2 shows stronger phenotypes)

  • Crosses with mutants in specific signaling pathways

• Treatment-specific responses:

  • ABA sensitivity assays (root growth, fresh weight)

  • Pathogen challenge tests

  • CEP peptide responses

• Cell-type specific analysis:

  • Focus on guard cells where CEPR2 is predominantly expressed

  • Separate analysis of stomatal versus systemic responses

How can CEPR2 antibodies be used to study receptor complex dynamics?

CEPR2 forms complexes with multiple proteins, presenting opportunities to study receptor complex dynamics:

• Temporal dynamics:

  • Time-course immunoprecipitation after stimulus application

  • Monitor complex assembly/disassembly kinetics

• Spatial dynamics:

  • Co-localization with interaction partners (CARs, PYLs, UBC34)

  • Membrane microdomain distribution

• Stimulus-dependent changes:

  • ABA treatment affects CEPR2-PYL interactions

  • CEP peptide binding may reorganize receptor complexes

• Post-translational modifications:

  • Phosphorylation state influences complex formation

  • Ubiquitination status affects receptor trafficking

• Complex composition analysis:

  • Sequential immunoprecipitation to isolate specific subcomplexes

  • Mass spectrometry to identify novel components

What methods can detect the phosphorylation state of CEPR2 itself?

As a kinase, CEPR2 is likely regulated by phosphorylation, though this aspect remains underexplored:

• Phospho-proteomic approaches:

  • Immunoprecipitate CEPR2 followed by mass spectrometry

  • Enrichment of phosphopeptides prior to analysis

• Mobility shift assays:

  • Phosphorylated proteins often migrate differently in SDS-PAGE

  • Phosphatase treatment to confirm phosphorylation-dependent shifts

• Phosphorylation site mutants:

  • Generate serine/threonine to alanine mutations

  • Assess functional consequences of preventing phosphorylation

• Phosphomimetic mutations:

  • Serine/threonine to aspartate/glutamate substitutions

  • Evaluate effects of constitutive "phosphorylation-like" state

• Phosphorylation-specific antibodies:

  • Development would require identification of key regulatory phosphosites

  • Could enable monitoring of CEPR2 activation state in vivo

How can CEPR2 antibodies contribute to understanding crosstalk between hormone signaling pathways?

CEPR2's involvement in both ABA responses and immunity positions it at a potential integration point for multiple signaling pathways:

• Co-immunoprecipitation under multiple hormone treatments:

  • Compare CEPR2 interactome after ABA, jasmonate, salicylic acid treatment

  • Identify common and hormone-specific interaction partners

• Receptor complex composition analysis:

  • Monitor how different hormones affect CEPR2-containing complexes

  • Track changes in phosphorylation patterns

• Subcellular localization changes:

  • Determine if hormones trigger CEPR2 endocytosis or relocalization

  • Co-localization with endosomal markers after stimulus

• Integration with other receptor systems:

  • Investigate potential interactions with other hormone receptors

  • Examine cross-phosphorylation between receptor families

• Temporal response dynamics:

  • Compare activation kinetics across different signaling pathways

  • Identify potential sequential activation patterns