Phospho-GSK3A/GSK3B (Y216 + Y279) Recombinant Monoclonal Antibody

What are GSK3A and GSK3B, and what is their role in cellular signaling?

GSK3A and GSK3B are two isoforms of Glycogen Synthase Kinase 3 (GSK3), a versatile serine/threonine kinase that is evolutionarily conserved across all eukaryotes. GSK3 is engaged in numerous cellular functions, from glycogen metabolism to cell cycle regulation and proliferation . It serves as a crucial regulator in multiple signaling pathways, including cellular responses to Wnt signaling, receptor tyrosine kinases, and G protein-coupled receptors . The two isoforms—GSK3α and GSK3β—have both distinct and overlapping functions in various cellular processes, allowing for nuanced regulation of downstream targets.

How does phosphorylation at Y216 (GSK3B) and Y279 (GSK3A) affect GSK3 activity?

Phosphorylation at tyrosine residues Y216 in GSK3β and Y279 in GSK3α activates the kinase activity of GSK3 . This tyrosine phosphorylation is considered an activating modification that enhances GSK3's ability to phosphorylate its downstream substrates. Unlike the inhibitory serine phosphorylation at positions S9/S21, the phosphorylation at these specific tyrosine residues serves as a marker for active GSK3 in research settings and is essential for maintaining the baseline catalytic activity of the enzyme.

What is the difference between tyrosine phosphorylation (Y216/Y279) and serine phosphorylation (S9/S21) of GSK3?

The key difference lies in their contrasting effects on GSK3 activity. Tyrosine phosphorylation at Y216 (GSK3β) and Y279 (GSK3α) activates GSK3 kinase activity, while serine phosphorylation at S9 (GSK3β) and S21 (GSK3α) inhibits GSK3 activity . The inhibitory serine-phosphorylation mechanism involves the creation of a pseudosubstrate that occupies the primed-substrate binding domain of GSK3, thereby preventing it from interacting with and phosphorylating its substrates . This dual regulatory mechanism allows for precise control of GSK3 activity in response to various cellular signals and creates a complex interplay between activation and inhibition pathways.

What cellular processes are regulated by GSK3A/GSK3B?

GSK3A/GSK3B regulates an extensive array of cellular processes including:

• Glycogen metabolism

• Cell cycle progression and proliferation

• Cell differentiation and development

• Apoptosis and cell survival

• Gene expression through transcription factor regulation

• Protein synthesis and degradation

• Cytoskeletal organization and cellular motility

In plants, GSK3-like kinases also play significant roles in developmental regulation and stress responses . GSK3 achieves this broad regulatory capacity by phosphorylating a diverse range of substrates including transcription factors, cofactors, kinases, scaffold proteins, cytoskeletal proteins, cyclins, metabolic enzymes, and components of the ubiquitin-proteasome system .

How do recombinant monoclonal antibodies differ from traditional monoclonal antibodies?

Recombinant monoclonal antibodies are produced using recombinant DNA technology, where the DNA sequence encoding the antibody is determined, cloned into an expression vector, and subsequently transfected into cell lines for expression . This differs from traditional monoclonal antibodies, which are typically produced using hybridoma technology. The production process for the Phospho-GSK3A/GSK3B (Y216 + Y279) recombinant antibody includes:

• Acquisition of the anti-GSK3 Beta-pY216 + GSK3 Alpha-pY279 monoclonal antibody using synthesized peptides

• Determination of the DNA sequence of the monoclonal antibody

• Cloning of the DNA sequence into a plasmid

• Transfection into cell lines for expression

• Purification using affinity-chromatography

This recombinant approach ensures consistent production, allows for specific modifications to enhance performance, and reduces batch-to-batch variability compared to traditional methods.

How can I validate the specificity of Phospho-GSK3A/GSK3B (Y216 + Y279) antibody in my experimental system?

A comprehensive validation strategy for Phospho-GSK3A/GSK3B (Y216 + Y279) antibody should include:

• Western blot analysis with known controls: Compare lysates from cells treated with modulators of GSK3 phosphorylation.

• Phosphatase treatment control: Treat a portion of your samples with lambda phosphatase to remove phosphate groups, which should eliminate antibody binding if it is truly phospho-specific.

• Gene silencing approaches: Use siRNA or CRISPR-Cas9 to knockdown GSK3A and GSK3B separately and together to confirm the specificity of the detected bands.

• Peptide competition assay: Pre-incubate the antibody with increasing concentrations of the phosphorylated peptide immunogen to demonstrate specific blocking of the signal.

• Cross-reactivity assessment: Test the antibody against recombinant phosphorylated and non-phosphorylated GSK3A and GSK3B proteins to confirm specificity for the phosphorylated forms.

• Immunohistochemistry (IHC) controls: Include positive control tissues known to express the phosphorylated forms and negative controls where the primary antibody is omitted.

• Multiple technique confirmation: Verify findings using complementary techniques such as mass spectrometry to identify phosphorylated residues.

This multi-faceted approach ensures the antibody specifically recognizes the phosphorylated Y216/Y279 epitopes of GSK3B/GSK3A, respectively.

For Immunohistochemistry (IHC):

• Recommended dilution range: 1:50-1:200

• Optimal fixation: 10% neutral buffered formalin

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Blocking: 1-5% BSA or normal serum from the same species as the secondary antibody

• Detection system: Streptavidin-biotin or polymer-based detection systems

• Counterstaining: Hematoxylin (light)

• Controls: Include both positive controls (tissues known to express phosphorylated GSK3) and negative controls

For ELISA:

• Starting dilution: Follow manufacturer's recommendations

• Coating buffer: Carbonate/bicarbonate buffer (pH 9.6)

• Blocking buffer: PBS with 1-5% BSA

• Sample preparation: Include phosphatase inhibitors

• Detection: HRP-conjugated secondary antibody with appropriate substrate

• Standard curve: Consider using recombinant phosphorylated GSK3 proteins

General Considerations:

• Always include phosphatase inhibitors in your lysis and sample preparation buffers

• Store antibody according to manufacturer recommendations (typically at -20°C or -80°C)

• Aliquot the antibody to avoid repeated freeze-thaw cycles that may reduce activity

• For optimal results, perform titration experiments to determine the ideal concentration for your specific sample type and application

How can I distinguish between GSK3A and GSK3B phosphorylation in my samples?

Distinguishing between GSK3A and GSK3B phosphorylation requires careful experimental design:

• Molecular weight discrimination: On Western blots, GSK3α (~51 kDa) and GSK3β (~47 kDa) can be distinguished by their different molecular weights when using the dual-specificity Phospho-GSK3A/GSK3B antibody.

• Isoform-specific antibodies: Use antibodies that specifically recognize either GSK3α or GSK3β in parallel with the phospho-specific antibody.

• Sequential immunoprecipitation: Perform immunoprecipitation with isoform-specific antibodies followed by Western blotting with the phospho-specific antibody.

• Genetic manipulation: Use siRNA or CRISPR-based approaches to selectively knockdown each isoform:

  • siRNA against GSK3α should reduce or eliminate the ~51 kDa band

  • siRNA against GSK3β should reduce or eliminate the ~47 kDa band

• Mass spectrometry: For definitive identification, use phospho-enrichment followed by mass spectrometry to identify isoform-specific phosphopeptides.

• Recombinant protein controls: Include purified recombinant GSK3α and GSK3β proteins (both phosphorylated and non-phosphorylated forms) as reference standards .

• Two-dimensional gel electrophoresis: Separate the isoforms based on both isoelectric point and molecular weight for enhanced discrimination.

What are the key considerations when designing experiments to study GSK3 substrate phosphorylation?

When designing experiments to study GSK3 substrate phosphorylation, consider:

• Substrate priming requirements: Most GSK3 substrates require priming phosphorylation following the S/T-X-X-X-S/T(P) motif, where GSK3 phosphorylates a serine/threonine four residues N-terminal to a pre-phosphorylated serine/threonine . Ensure your experimental design accounts for the activity of relevant priming kinases.

• Spatial and temporal coordination: Both signals (GSK3 activation and substrate priming) must coincide temporally and spatially in your experimental system .

• Substrate structure: The canonical four-residue spacing between the primed site and GSK3 target site can vary based on the three-dimensional structure of the substrate . Consider this when identifying potential GSK3 targets.

• Non-primed substrates: Some GSK3 substrates don't require priming. For these substrates, inhibitory serine-phosphorylation of GSK3 may not regulate their phosphorylation .

• Sequential phosphorylation: Many GSK3 substrates contain multiple sequential target sites, creating a hierarchical phosphorylation pattern. For example, GSK3 phosphorylates serines 652, 648, 644, and 640 in glycogen synthase .

• Sample preparation: Include phosphatase inhibitors in all buffers to preserve phosphorylation status.

• Controls: Include both positive controls (known GSK3 substrates like glycogen synthase) and negative controls (substrates with mutated GSK3 target sites).

• Kinase assays: Consider in vitro kinase assays with purified components to confirm direct GSK3-mediated phosphorylation versus indirect effects.

How does GSK3 substrate priming influence the interpretation of results when using this antibody?

GSK3 substrate priming significantly impacts result interpretation because:

• Dual signal requirement: For most GSK3 substrates, two signals must coincide: GSK3 activation (which Phospho-GSK3A/GSK3B (Y216 + Y279) antibody helps monitor) and substrate priming by another kinase .

• Priming-dependent phosphorylation: Changes in substrate phosphorylation may result from alterations in either GSK3 activity or priming kinase activity. Increased Y216/Y279 phosphorylation might not translate to increased substrate phosphorylation if priming is limiting.

• Hierarchical phosphorylation: Many GSK3 substrates contain multiple sequential phosphorylation sites. After the initial priming phosphorylation, GSK3 can create its own primed substrate in a stepwise fashion, phosphorylating every fourth residue in a string of sequential sites .

• Non-primed substrates: Some GSK3 substrates don't require priming phosphorylation and may be regulated differently than primed substrates .

• Experimental design implications: To properly interpret results:

  • Monitor both GSK3 phosphorylation status and the priming status of your substrate

  • Include controls that manipulate priming kinases as well as GSK3

  • Consider time-course experiments to capture the sequential nature of the phosphorylation events

  • For comprehensive analysis, use phospho-specific antibodies that recognize both the GSK3 target site and the priming site

• Pathway cross-talk: Priming kinases (often casein kinase 1, CDKs, or MAPKs) represent points of cross-talk between GSK3 and other signaling pathways that must be considered when interpreting results.

Positive Controls:

• Cell/tissue treatment:

  • Treatment with Wnt pathway inhibitors (which may increase GSK3 activity)

  • Neuronal cells with constitutively high levels of GSK3 tyrosine phosphorylation

  • Specific cancer cell lines with elevated GSK3 activity

• Recombinant proteins:

  • Purified GSK3α or GSK3β that has been phosphorylated at Y279 or Y216, respectively

  • In vitro phosphorylated GSK3 using purified tyrosine kinases

• Known substrate phosphorylation:

  • Monitor phosphorylation of well-characterized GSK3 substrates (e.g., glycogen synthase, β-catenin) as functional validation

Negative Controls:

• Phosphatase treatment:

  • Samples treated with lambda phosphatase to remove phosphate groups, eliminating antibody binding

• Genetic approaches:

  • GSK3α and GSK3β knockdown or knockout samples

  • Cells expressing GSK3 with Y279A or Y216A mutations that cannot be phosphorylated at these sites

• Peptide competition:

  • Pre-incubation of the antibody with the phosphopeptide immunogen to block specific binding

• Antibody controls:

  • Isotype control antibody (same species and isotype but irrelevant specificity)

  • Omission of primary antibody in immunostaining procedures

• Cross-reactivity assessment:

  • Testing against non-phosphorylated GSK3 to confirm phospho-specificity

Using these controls in combination provides robust validation of antibody specificity and experimental results.

How can I correlate GSK3 Y216/Y279 phosphorylation with its kinase activity in my research?

To establish correlation between GSK3 Y216/Y279 phosphorylation and kinase activity:

• Parallel assessment: Measure both phosphorylation status (using the Phospho-GSK3A/GSK3B antibody) and kinase activity in the same samples.

• Kinase activity assay: Perform in vitro kinase assays using:

  • Immunoprecipitated GSK3 from your samples

  • Appropriate substrates such as phosphoglycogen synthase peptide-2

  • Reaction buffer containing MgCl₂, ATP, and [γ-³²P] ATP

  • Analysis by spotting reactions onto phosphocellulose paper and measuring radioactivity

• Manipulation of phosphorylation: Use tyrosine kinase inhibitors or activators to modulate Y216/Y279 phosphorylation and observe corresponding changes in kinase activity.

• Phosphomimetic and phosphodeficient mutants: Compare activity of:

  • Wild-type GSK3

  • Y216E/Y279E phosphomimetic mutants

  • Y216F/Y279F phosphodeficient mutants

• Monitor endogenous substrate phosphorylation: Examine phosphorylation status of well-characterized GSK3 substrates like glycogen synthase or β-catenin as functional readouts.

• Consider inhibitory serine phosphorylation: Simultaneously monitor both tyrosine (Y216/Y279) and serine (S9/S21) phosphorylation, as the inhibitory serine phosphorylation can override the activating effect of tyrosine phosphorylation .

• Time-course studies: Track changes in both phosphorylation and activity over time following cellular stimulation to establish temporal relationships.

This multi-faceted approach will provide robust evidence for the correlation between phosphorylation status and functional activity.

What are the potential cross-talk mechanisms between GSK3 and other signaling pathways that could affect my results?

GSK3 is integrated into multiple signaling networks with extensive cross-talk potential:

• Wnt signaling pathway:

  • Directly regulates GSK3 activity toward β-catenin without affecting global GSK3 activity

  • Forms a destruction complex with Axin, APC, and CK1

  • May sequester GSK3 in multivesicular bodies upon Wnt activation

• Insulin/growth factor signaling:

  • PI3K/AKT pathway phosphorylates GSK3 at inhibitory serines (S21/S9)

  • Creates feedback loops as GSK3 can phosphorylate insulin receptor substrate-1 (IRS-1)

• MAPK pathway interactions:

  • MAPKs can phosphorylate the same substrates as GSK3 (e.g., SPCH in plants)

  • MAPKs can serve as priming kinases for GSK3 substrates

• PKA signaling:

  • PKA can phosphorylate and modulate GSK3 activity

  • Creates potential for cAMP-dependent regulation of GSK3 function

• Priming kinase cross-talk:

  • Casein kinase 1 (CK1), CDKs, or MAPKs often act as priming kinases

  • Activity of these kinases directly impacts GSK3's ability to phosphorylate primed substrates

• Transcription factor regulation:

  • GSK3 phosphorylates numerous transcription factors (e.g., MYBL2, AIF2, ABI5, UPB1)

  • Creates feedback loops in various signaling networks

• Cellular stress responses:

  • p38 MAPK and JNK pathways intersect with GSK3 signaling during stress responses

  • GSK3 can modulate cellular responses to oxidative stress and DNA damage

When designing experiments, include controls that help distinguish direct GSK3 effects from indirect consequences of cross-talk, and consider time-course experiments to resolve the sequence of events in complex signaling cascades.

How can I effectively use this antibody to study GSK3's role in specific disease models?

To effectively use Phospho-GSK3A/GSK3B (Y216 + Y279) antibody in disease models:

• Neurodegenerative disease models:

  • Perform IHC at 1:50-1:200 dilution to compare phosphorylation patterns between affected and unaffected brain regions

  • Correlate with pathological markers (e.g., tau phosphorylation, amyloid plaques)

  • Examine temporal changes during disease progression

• Cancer models:

  • Compare phosphorylation levels between tumor and adjacent normal tissue

  • Correlate with proliferation markers, invasion, and metastasis potential

  • Examine changes before and after treatment with targeted therapies

• Metabolic disorder models:

  • Analyze tissue-specific changes in GSK3 phosphorylation (liver, muscle, adipose)

  • Correlate with glycogen synthase activity and insulin signaling components

  • Monitor changes in response to diet, exercise, or pharmacological interventions

• Experimental design considerations:

  • Establish baseline phosphorylation patterns in your model system under normal conditions

  • Include appropriate controls (age-matched, vehicle-treated, genetic background)

  • Use multiple techniques to confirm findings (IHC, Western blot, activity assays)

• Validation approaches:

  • Pharmacological modulation using GSK3 inhibitors (e.g., lithium, CHIR99021)

  • Genetic approaches (conditional knockouts, knockdowns, or overexpression)

  • Correlation with known GSK3 substrates relevant to the disease model

• Translational considerations:

  • Complement animal model studies with analysis of human patient samples

  • Consider tissue microarrays for high-throughput analysis across multiple patient samples

  • Correlate molecular findings with clinical parameters and outcomes

This comprehensive approach will help establish meaningful connections between GSK3 phosphorylation status and disease pathophysiology.

What techniques can be combined with this antibody to comprehensively analyze GSK3 signaling dynamics?

For comprehensive analysis of GSK3 signaling dynamics, combine multiple complementary techniques:

• Phospho-specific flow cytometry:

  • Analyze GSK3 phosphorylation at the single-cell level

  • Reveal population heterogeneity in response to treatments

  • Combine with other signaling markers for multiplex analysis

• Proximity ligation assay (PLA):

  • Visualize interactions between phosphorylated GSK3 and its substrates in situ

  • Detect protein-protein interactions with spatial resolution

  • Quantify changes in interactions following stimulation or inhibition

• Phosphoproteomics:

  • Use mass spectrometry to simultaneously monitor GSK3 phosphorylation and global phosphoproteome changes

  • Identify novel GSK3 substrates with the characteristic phosphorylation motif

  • Quantify changes in substrate phosphorylation following GSK3 modulation

• FRET-based biosensors:

  • Monitor GSK3 activity in real-time in living cells

  • Track spatiotemporal dynamics of signaling events

  • Observe subcellular localization of active GSK3

• Chromatin immunoprecipitation (ChIP):

  • Identify genomic regions affected by GSK3-regulated transcription factors

  • Combine with RNA-seq to correlate binding events with gene expression changes

  • Track dynamic changes in transcriptional regulation

• In vitro kinase assays:

  • Directly measure GSK3 kinase activity using purified components

  • Test substrate specificity and the effects of inhibitors

  • Compare activity of differentially phosphorylated GSK3 forms

• Multi-label immunohistochemistry/immunofluorescence:

  • Simultaneously detect multiple components of the GSK3 signaling pathway

  • Analyze cellular and subcellular co-localization

  • Perform at recommended dilutions of 1:50-1:200

• Live-cell imaging:

  • Track dynamic changes in GSK3 localization and activity

  • Monitor substrate phosphorylation in real-time

  • Observe cellular responses to perturbations in GSK3 signaling

This multi-technique approach provides a comprehensive view of GSK3 signaling dynamics across different levels of biological organization.