Phospho-CREB1 (Ser142) Antibody

Advanced Research Questions

• How can I validate the specificity of my Phospho-CREB1 (Ser142) antibody?

  Validating antibody specificity is critical for reliable data interpretation. For Phospho-CREB1 (Ser142) antibodies, implement these methodological approaches:

  1. Peptide competition assay: Pre-incubate the antibody with the phosphopeptide used as the immunogen (sequence around phosphorylation site of Ser142) . A specific antibody will show reduced or eliminated signal.

  2. Phosphatase treatment: Treat half of your sample with lambda phosphatase before Western blotting. A phospho-specific antibody should show diminished signal in the treated sample.

  3. Genetic validation: Use lysates from cells expressing dominant negative CREB with serine-to-alanine mutations at Ser142 (CREBdn-S142A) . The antibody should show reduced signal compared to wild-type CREB.

  4. Stimulus-dependent phosphorylation: Compare samples from resting cells versus stimulated cells (e.g., KCl depolarization for neurons) . The antibody should detect increased phosphorylation in stimulated samples.

  5. Cross-reactivity assessment: Test the antibody against related phosphoproteins or other CREB family members to ensure specificity.

  Some commercial Phospho-CREB1 (Ser142) antibodies are purified using affinity-chromatography with epitope-specific phosphopeptides, with non-phospho specific antibodies removed by chromatography using non-phosphopeptides . This purification strategy enhances specificity but still requires experimental validation.

• What experimental approaches are optimal for studying the functional consequences of CREB phosphorylation at Ser142 in neuronal plasticity?

  To investigate functional consequences of CREB phosphorylation at Ser142 in neuronal plasticity, consider these methodological approaches:

  1. Viral vector-mediated expression of mutant CREB: Use herpes simplex virus (HSV) constructs containing GFP-tagged dominant-negative CREB with serine-to-alanine mutations at Ser142/143 (CREBdn-S142A/S143A) . This approach allows for specific blockade of phosphorylation at these sites.

  2. In vivo electrophysiology: Implement visual evoked potential (VEP) recordings in virus-injected animals before and after monocular deprivation to assess ocular dominance plasticity . This approach allows for within-subject comparisons of neuronal responses.

  3. Immediate early gene expression analysis: Measure activity-dependent expression of genes like Arc, which has been shown to require Ser142/143 phosphorylation . Use both immunocytochemistry and Western blotting for comprehensive analysis.

  4. Calcium imaging: Since Ser142/143 phosphorylation is regulated by calcium-dependent mechanisms, combine phosphorylation studies with calcium imaging to correlate calcium dynamics with CREB phosphorylation states.

  5. Transcriptomic profiling: Compare gene expression profiles between neurons expressing wild-type CREB versus CREBdn-S142A/S143A to identify the specific gene programs regulated by Ser142/143 phosphorylation .

  When designing these experiments, it's crucial to include appropriate controls, such as neurons expressing GFP alone and neurons expressing CREBdn-S133A, to distinguish the specific effects of Ser142/143 phosphorylation from those of Ser133 phosphorylation .

• How can I distinguish between Ser142 and Ser143 phosphorylation effects when they commonly occur together?

  Distinguishing between the individual contributions of Ser142 and Ser143 phosphorylation requires sophisticated experimental strategies:

  1. Single-site mutants: Generate separate Ser142Ala and Ser143Ala mutants, in addition to the double mutant. Compare phenotypes to identify distinct effects.

  2. Phospho-specific antibodies: While challenging, develop or source antibodies that specifically recognize only Ser142 or only Ser143 phosphorylation. The literature suggests this is difficult as Ser143 phosphorylation detection typically requires prior Ser142 phosphorylation .

  3. Mass spectrometry: Implement phospho-proteomics approaches with high-resolution mass spectrometry to quantitatively measure the stoichiometry of phosphorylation at each site separately.

  4. Temporal dynamics: Design time-course experiments to determine if phosphorylation occurs sequentially rather than simultaneously. Some studies suggest Ser142 phosphorylation may precede Ser143 .

  5. Kinase manipulation: Identify and selectively inhibit the kinases responsible for phosphorylation at each site. Previous research has shown that different stimuli and signaling pathways might preferentially activate phosphorylation at specific sites.

  Current evidence suggests that phosphorylation at Ser142 and Ser143 works in concert, with Ser142 phosphorylation possibly being a prerequisite for Ser143 phosphorylation . This hierarchical relationship makes it particularly challenging but important to design experiments that can distinguish their individual contributions.

• What are the recommended protocols for double immunostaining to detect both pCREB(Ser133) and pCREB(Ser142) in the same samples?

  Double immunostaining for pCREB(Ser133) and pCREB(Ser142) requires careful protocol optimization:

  Step	Protocol Details
  Fixation	4% paraformaldehyde for 20 minutes at room temperature
  Washing	PBS with 100 mM glycine (PBS/Gly)
  Blocking	10% normal goat serum (NGS) in PBS/Gly with 0.1% Triton X-100 for 60 min at 37°C
  Primary antibodies	Use rabbit anti-pCREB Ser133 (1:1000, Millipore) and rabbit anti-pCREB Ser142/143 (1:500) from different host species
  Incubation	4°C overnight in PBS/Gly/0.1% Triton X-100 in 5% NGS
  Secondary antibodies	Species-specific fluorescent antibodies with non-overlapping emission spectra
  Counterstaining	Include CaMKII as an excitatory neuronal marker (1:500, Abcam)
  Mounting	Permafluor mountant (Thermo Scientific)

  Since both phospho-specific antibodies are often raised in rabbits, consider these strategies:

  1. Sequential immunostaining: Complete the first staining with one antibody, followed by an additional fixation step, then proceed with the second antibody.

  2. Direct conjugation: Directly conjugate one antibody with a fluorophore to eliminate the need for secondary antibody.

  3. Zenon labeling: Use Zenon technology to pre-label one of the rabbit antibodies with a fluorescent Fab fragment.

  4. Alternative approach: Instead of double staining, use consecutive sections or split your samples for parallel single staining if the above methods yield high background.

  For optimal results, include appropriate controls including single-stained samples and phosphatase-treated negative controls.

• How should I design experiments to investigate the relationship between neuronal activity, calcium signaling, and CREB phosphorylation at Ser142?

  To investigate the relationship between neuronal activity, calcium signaling, and CREB Ser142 phosphorylation:

  1. Stimulation protocols: Design experiments with varying stimulation paradigms:

     • High KCl (50 mM) for membrane depolarization

     • Glutamate receptor agonists (NMDA, AMPA)

     • Synaptic stimulation protocols (theta-burst, high-frequency)

     • Optogenetic stimulation for cell-type specific activation

  2. Calcium manipulation: Systematically alter calcium dynamics:

     • Use calcium chelators (BAPTA-AM) to block calcium signaling

     • Employ calcium ionophores to increase intracellular calcium

     • Manipulate calcium channel activity (L-type, T-type inhibitors)

     • Utilize calcium imaging (GCaMP) to correlate calcium transients with CREB phosphorylation

  3. Kinase pathway analysis: Investigate the calcium-dependent kinases involved:

     • CaMKII/IV inhibitors (KN-93, KN-62)

     • CaMK genetic knockdowns or dominant-negative constructs

     • Combinatorial inhibition of multiple kinase pathways

  4. Temporal analysis: Implement time-course experiments:

     • Short-term (seconds to minutes) to capture initial phosphorylation events

     • Long-term (hours) to study maintenance of phosphorylation

     • Correlation with immediate early gene expression (Arc)

  5. Single-cell resolution: Use immunocytochemistry with nuclear and cytoplasmic markers to assess:

     • Subcellular localization of phosphorylated CREB

     • Correlation between CaMKII activation and CREB phosphorylation

     • Cell-type specific responses using markers like CaMKII for excitatory neurons

  Previous research has demonstrated that KCl stimulation increases nuclear staining of both pCREB(Ser133) and pCREB(Ser142/143) compared to control conditions , providing a baseline protocol for these investigations.

• What are the methodological considerations for using phospho-CREB antibodies in brain tissue from disease models?

  When using phospho-CREB(Ser142) antibodies in disease model brain tissue, consider these methodological approaches:

  1. Tissue preservation:

     • Rapid post-mortem fixation is critical as phosphorylation states degrade quickly

     • Perfusion fixation with 4% paraformaldehyde for animal models

     • Flash freezing for biochemical analyses to preserve phosphorylation

     • Use of phosphatase inhibitor cocktails in all buffers

  2. Sectioning considerations:

     • For IHC, optimal thickness is 30-40 μm for floating sections

     • For IF, thinner sections (10-20 μm) may provide better resolution

     • Antigen retrieval methods may need optimization for phospho-epitopes

  3. Controls and validation:

     • Age-matched, treatment-matched controls are essential

     • Include positive controls (e.g., tissue from animals following seizure induction)

     • Negative controls via phosphatase treatment of select sections

     • Quantify total CREB in parallel sections for normalization

  4. Quantification methods:

     • Use digital image analysis with standardized thresholding

     • Employ cell counting for nuclear localization of phospho-CREB

     • Consider laser scanning cytometry for high-throughput analysis

     • Normalize phospho-CREB to total CREB when possible

  5. Disease-specific considerations:

     • In neurodegenerative conditions, account for cell loss when interpreting results

     • For seizure or excitotoxicity models, compare affected vs. unaffected regions

     • In developmental disorders, consider age-dependent changes in CREB expression

     • For psychiatric models, correlate with behavioral endpoints

  When reporting findings, clearly describe fixation protocols, antibody dilutions (typically 1:100-1:300 for IHC) , and quantification methods to ensure reproducibility and reliable interpretation of disease-related changes in CREB phosphorylation.

• How can I integrate pCREB(Ser142) analysis with transcriptomic approaches to identify specific gene programs regulated by this phosphorylation site?

  To integrate pCREB(Ser142) analysis with transcriptomics for identifying regulated gene programs:

  1. Experimental design:

     • Compare gene expression profiles between wild-type and CREBdn-S142A/S143A expressing neurons

     • Include CREBdn-S133A as a comparative control to distinguish Ser142/143-specific programs

     • Design time-course experiments to capture immediate early, intermediate, and late-response genes

     • Combine with stimulus-specific conditions (e.g., KCl, neurotrophins, synaptic activity)

  2. ChIP-seq approach:

     • Perform chromatin immunoprecipitation with pCREB(Ser142) antibodies

     • Create binding profiles at different time points after stimulation

     • Integrate with transcriptomic data to correlate binding with expression changes

     • Identify enriched DNA motifs in Ser142-dependent target genes

  3. Cell-type specific analysis:

     • Use FACS or single-cell approaches to isolate specific neuronal populations

     • Compare pCREB(Ser142)-dependent transcriptomes across cell types

     • Correlate with cell-type specific functional outcomes

  4. Bioinformatic analysis pipeline:

     • Identify significantly changed genes (FDR < 0.05, fold-change > 1.5)

     • Perform Gene Ontology and pathway enrichment analyses

     • Compare with existing CREB target databases

     • Develop prediction models for Ser142-specific gene regulatory networks

  5. Validation strategies:

     • Confirm select targets with qRT-PCR and protein analysis

     • Use luciferase reporter assays with wild-type and mutant CREB binding sites

     • Employ CRISPR-based approaches to manipulate identified regulatory regions

     • Correlate with functional outcomes like neuronal plasticity measures

  Research has identified Arc as a gene requiring pCREB(Ser142/143) for its expression , providing a valuable positive control for these studies. This integrated approach will help distinguish the specific gene programs regulated by different CREB phosphorylation patterns, advancing our understanding of the "CREB code" in neuronal function.