AKAP10 Antibody

What is AKAP10 and why is it significant in cellular signaling research?

AKAP10 (A-kinase anchoring protein 10) is a member of the AKAP family that plays a crucial role in cellular signaling by anchoring protein kinase A (PKA) to specific subcellular locations. This spatial regulation is essential for compartmentalized signaling responses.

AKAP10 is particularly significant because:

• It is a dual-specific AKAP, interacting with both type I and type II regulatory subunits of PKA

• It is highly enriched in mitochondria and contains RGS domains in addition to PKA-binding domains

• It functions in G-protein coupled signal transduction pathways

• It serves as an adaptor in the assembly of multiprotein complexes that regulate various cellular processes, including apoptosis

The biological relevance of AKAP10 extends to heart rhythm regulation, immune responses, and potential involvement in cancer progression, making AKAP10 antibodies valuable tools in multiple research areas .

What are the common applications for AKAP10 antibodies in research?

AKAP10 antibodies have been validated for multiple experimental applications:

When using AKAP10 antibodies in IHC applications, antigen retrieval is often suggested with TE buffer (pH 9.0) or citrate buffer (pH 6.0) . For optimal results, each antibody should be titrated in the specific experimental system .

How should I select the appropriate AKAP10 antibody for my experiment?

Selection of an AKAP10 antibody should be based on several critical factors:

• Target species compatibility: Verify that the antibody recognizes AKAP10 in your species of interest. While many antibodies react with human AKAP10, reactivity with mouse or rat AKAP10 varies between products .

• Epitope location: Consider which region of AKAP10 the antibody recognizes. Some antibodies target the N-terminal region , while others target the C-terminal region or middle sections. This is particularly important if:

  • You're studying specific AKAP10 isoforms

  • Your research involves SNPs or mutations in specific regions

  • You need to distinguish between nuclear and mitochondrial AKAP10 variants

• Validated applications: Ensure the antibody has been validated for your specific application. For example, not all AKAP10 antibodies that work in Western blotting will perform well in immunofluorescence .

• Clonality consideration:

  • Polyclonal antibodies offer broader epitope recognition but may have more batch-to-batch variation

  • Monoclonal antibodies provide higher specificity to a single epitope and more consistency between lots

• Host species compatibility: Choose an antibody from a host species that won't conflict with other antibodies in multiplexed experiments.

For rigorous validation, consider testing multiple antibodies targeting different epitopes to confirm specificity, particularly when studying novel aspects of AKAP10 biology .

What controls should be included when working with AKAP10 antibodies?

Proper experimental controls are essential for meaningful interpretation of AKAP10 antibody experiments:

Positive Controls:

• Cell lines with confirmed AKAP10 expression (HeLa, HEK-293, and Jurkat cells have been validated)

• Tissues with known AKAP10 expression (heart, brain, testis, skeletal muscle)

• Recombinant AKAP10 protein (when available)

Negative Controls:

• Primary antibody omission

• Isotype control (same isotype as the primary antibody but with irrelevant specificity)

• For genetic studies: AKAP10 knockdown or knockout samples when available

• Blocking peptide experiments to confirm specificity

Validation Controls:

• When studying AKAP10 variants or isoforms, include wild-type AKAP10 samples for comparison

• When investigating subcellular localization, include co-staining with established organelle markers (particularly mitochondrial markers)

• For functional studies, consider using AKAP-PKA disrupting peptides (such as st-Ht31 or RIAD) as controls to confirm AKAP-mediated effects

Technical Considerations:

• Include molecular weight markers for Western blots (AKAP10 typically appears at 74 kDa)

• For IHC/IF, include appropriate blocking of endogenous peroxidases or fluorescence

• When quantifying AKAP10 expression in tissues, use standardized scoring systems (0-3 scale has been used in previous studies)

How can AKAP10 antibodies be used to study SNP-related structural and functional changes?

Single nucleotide polymorphisms (SNPs) in AKAP10 have been associated with altered protein function and disease susceptibility. AKAP10 antibodies can be valuable tools for studying these variants through several approaches:

• Differential binding analysis: Using antibodies targeting different epitopes may reveal structural changes caused by SNPs. For example, the I646V polymorphism alters AKAP10's affinity for PKA regulatory subunits, which might be detectable using specific antibodies .

• Proximity ligation assays: Combining AKAP10 antibodies with antibodies against PKA regulatory subunits in proximity ligation assays can reveal how SNPs affect protein-protein interactions in situ.

• Subcellular localization studies: The V282M variant in AKAP18γ (an AKAP10 isoform) affects nuclear localization. Immunofluorescence with appropriate AKAP10 antibodies can detect such mislocalization phenomena .

• Co-immunoprecipitation protocols: To assess how SNPs affect AKAP10's interactions with PKA and other binding partners:

  • Use AKAP10 antibodies for immunoprecipitation followed by Western blotting for interacting proteins

  • Compare wild-type and SNP variant proteins using antibodies that recognize shared epitopes

• Functional correlation studies: Combine AKAP10 antibody detection with functional readouts such as:

  • Ca²⁺ current measurements in cardiac cells (affected by AKAP10 variants)

  • cAMP responses in airway smooth muscle cells

  • TLR4-mediated inflammatory responses

When studying the I646V polymorphism specifically, researchers should note that it affects PKA binding affinity and has been associated with cardiac phenotypes including heart rate regulation and heart rate variability in human populations .

What approaches can resolve conflicting AKAP10 antibody data in cancer research?

Research on AKAP10's role in cancer has produced varying results. When antibody data shows inconsistencies, consider these methodological approaches:

• Isoform-specific analysis:

  • AKAP10 has multiple splice variants including nuclear-specific isoforms (e.g., AKAP18ε)

  • Use RT-PCR with isoform-specific primers to determine which variants are expressed

  • Select antibodies that can distinguish between isoforms or target common regions

• Technical validation across methods:

  • Combine antibody-based detection (IHC/WB) with mRNA analysis (qPCR/RNA-seq)

  • Use multiple antibodies targeting different epitopes

  • Employ genetic approaches (siRNA knockdown) to confirm specificity

• Context-dependent interpretation:

  • AKAP10 expression in colorectal cancer has been reported to correlate with tumor invasion, lymph node metastasis, and differentiation status

  • Contradictions might reflect true biological variation rather than technical issues

  • Stratify analyses by cancer subtype, stage, and molecular characteristics

• Correlation with genotype:

  • Document the AKAP10 2073A/G polymorphism status in samples

  • Research shows variant genotypes correlate with increased AKAP10 expression in colorectal cancer (68% positive expression in variant genotypes vs. 52% in AA genotype)

Resolution protocol for contradictory findings:

• Score AKAP10 immunostaining using standardized criteria (0-3 scale has been validated)

• Document subcellular localization carefully (nuclear vs. mitochondrial)

• Correlate findings with clinicopathological data

• Consider tumor heterogeneity by analyzing multiple regions when possible

How can AKAP10 antibodies be utilized in studies of PKA signaling compartmentalization?

AKAP10's function in PKA localization makes it a valuable target for studying compartmentalized signaling:

• Super-resolution microscopy approaches:

  • Combine AKAP10 antibodies with PKA subunit antibodies for STORM or STED microscopy

  • This can reveal nanoscale organization of signaling complexes

  • Include phospho-specific antibodies for PKA substrates to map active signaling domains

• Live-cell FRET-based analysis:

  • Use fluorescently tagged antibody fragments with PKA activity biosensors

  • This approach can monitor real-time changes in PKA activity in AKAP10-positive compartments

• Differential centrifugation coupled with immunoblotting:

  • Separate subcellular fractions (cytosolic, nuclear, mitochondrial)

  • Use AKAP10 antibodies to track distribution across fractions

  • Correlate with PKA catalytic and regulatory subunit distribution

• AKAP-PKA disruption studies:

  • Use st-Ht31 or RIAD peptides to disrupt AKAP-PKA interactions

  • Monitor changes in downstream signaling using phospho-specific antibodies

  • Compare to targeted AKAP10 knockdown effects

• Cross-linking approaches:

  • Chemical cross-linking followed by AKAP10 immunoprecipitation

  • Mass spectrometry analysis to identify components of AKAP10 signaling complexes

  • This has revealed novel interaction partners beyond PKA subunits

Research should consider the dual specificity of AKAP10, which can bind both type I and type II regulatory subunits of PKA, a relatively uncommon feature among AKAPs .

What methodological adaptations are needed when using AKAP10 antibodies in respiratory tissue research?

Respiratory tissues present unique challenges for AKAP10 antibody applications that require specific methodological considerations:

• Fixation and antigen retrieval optimization:

  • Respiratory tissues often require specialized fixation due to their air-filled structure

  • For AKAP10 detection in lung tissues, 4% paraformaldehyde fixation for 24h has shown good results

  • Antigen retrieval with citrate buffer (pH 6.0) may better preserve airway smooth muscle (ASM) structure than TE buffer

• Cell-type specific analysis:

  • Respiratory tissues contain diverse cell populations with potentially different AKAP10 expression

  • Use cell-type specific markers in combination with AKAP10 antibodies:

    • α-smooth muscle actin for ASM

    • Surfactant proteins for alveolar epithelial cells

    • CD68 for alveolar macrophages

• Expression quantification approaches:

  • Semiquantitative evaluation using a 0-3 scale has been validated for AKAP proteins in respiratory tissues

  • Classification: 0 (no staining), 1 (low), 2 (medium), and 3 (strong)

  • Score both ASM-specific and total tissue staining separately

• Functional correlation studies:

  • AKAP proteins coordinate inflammatory responses to cigarette smoke extract (CSE) in ASM cells

  • When studying AKAP10 in respiratory inflammation, correlate antibody staining with:

    • IL-8 release measurements

    • cAMP responses

    • Phosphorylation of downstream targets like VASP and ERK

• Disease-specific considerations:

  • In asthma and COPD research, consider how β2-adrenoceptor desensitization affects AKAP10 function

  • Document patient medication status (β2-agonists, theophylline, muscarinic antagonists) as these affect AKAP-mediated signaling

How should experiments be designed to investigate AKAP10's role in innate immunity?

AKAP10 has emerging roles in innate immune responses that can be investigated using these experimental approaches:

• Toll-like receptor (TLR) pathway analysis:

  • AKAP10 mediates TLR4-induced nitric oxide production and cytokine responses

  • Design experiments to measure:

    • iNOS induction (by WB, qPCR)

    • NO production (Griess assay)

    • IL-10 and IL-6 production (ELISA)

    • Activation states of NF-κB and CREB (using phospho-specific antibodies)

• Macrophage activation studies:

  • In alveolar macrophages, AKAP10 shapes TLR responses to lipopolysaccharide (LPS)

  • Protocol design should include:

    • Dose-response curves for LPS stimulation

    • Time-course analysis of AKAP10 expression after stimulation

    • Correlation with PGE2 production

• AKAP10-PKA complex disruption:

  • Use RII/AKAP disrupter peptide Ht31 to attenuate IL-10 and IL-6 production

  • Use RI/AKAP disrupter peptide (RIAD) to attenuate NO production

  • Include appropriate peptide controls

• Genetic manipulation approaches:

  • siRNA knockdown of AKAP10 in macrophage cell lines

  • CRISPR/Cas9 editing to study specific domains

  • Comparison with other AKAPs (AKAP11 is involved in IL-6 production)

• Co-immunoprecipitation studies:

  • Use AKAP10 antibodies to pull down complexes before and after TLR stimulation

  • Identify dynamic changes in the AKAP10 interactome during immune activation

  • Focus on interactions with PKA regulatory subunits and components of the TLR signaling pathway

When designing these experiments, consider that TNF-α generation is mediated by PGE2, which is believed to involve PKARII-AKAP complexes . Document the timing of these responses carefully, as the temporal dynamics of AKAP10-mediated signaling in immune cells may differ from other systems.

What protocols are recommended for investigating AKAP10 splice variants and isoforms?

The AKAP10 gene produces multiple transcripts and protein isoforms with distinct functions. To study these variants:

• Isoform identification strategy:

  • Review databases like TCGA SpliceSeq to identify known and predicted AKAP10 splice variants

  • Design PCR primers spanning exon junctions to amplify specific isoforms

  • Example: AKAP18ε, a non-PKA-anchoring nuclear isoform, can be detected using primers that bridge sequences in exon 9 and exon 12.2

• Antibody selection for isoform discrimination:

  • Choose antibodies with epitopes in isoform-specific regions

  • When epitope information is limited, validate antibodies against recombinant isoforms

  • Consider using panels of antibodies targeting different regions

• Subcellular localization protocol:

  • Use immunofluorescence with confocal microscopy

  • Include co-localization with organelle markers:

    • Nuclear markers (e.g., DAPI) for AKAP18ε

    • Mitochondrial markers for canonical AKAP10

  • Example finding: AKAP18ε-GFP localizes exclusively to the nucleus while RIIα and α-tubulin remain cytoplasmic

• Functional characterization:

  • Immunoprecipitation to determine binding partners of each isoform

  • For AKAP18ε, which lacks PKA-binding capacity, focus on identifying non-PKA binding partners

  • Enzyme-mediated proximity-proteomics to discover compartment-specific interaction partners

• Expression profiling across tissues:

  • RT-qPCR with isoform-specific primers

  • RNA-seq analysis with appropriate splice-aware alignment

  • Tissue microarrays with isoform-specific antibodies

The discovery of AKAP18ε, which lacks a PKA-anchoring helix, demonstrates that not all AKAP10 isoforms function as PKA-anchoring proteins . This has profound implications for interpreting AKAP10 localization and function data.

How can AKAP10 antibodies be employed in cardiovascular disease research?

AKAP10 plays significant roles in cardiac function, particularly in heart rhythm regulation. Research approaches include:

• Genetic correlation studies:

  • The human AKAP10 I646V polymorphism affects PKA binding and correlates with cardiac phenotypes

  • Protocol for genotype-phenotype correlation:

    • Genotype samples for I646V polymorphism

    • Use AKAP10 antibodies to assess protein levels and localization

    • Correlate with heart rate and heart rate variability measurements

• Cardiac cell experiments:

  • In embryonic stem cell-derived cardiac myocytes, AKAP10 mutations affect contraction rate

  • Experimental design should include:

    • Immunofluorescence with AKAP10 antibodies to confirm localization

    • Treatment with cholinergic agonists (carbachol) and adrenergic agonists

    • Measurement of contraction rates and beat-to-beat variability

• Vagus nerve sensitivity assessment:

  • AKAP10 regulates cardiac response to vagus nerve input

  • In animal models, phenylephrine-induced baroreflexes can be measured

  • In human studies, 24-hour ECG monitoring with heart rate variability analysis

• Molecular mechanism investigation:

  • Combine AKAP10 antibodies with phospho-specific antibodies for PKA targets

  • Focus on calcium-handling proteins in cardiomyocytes

  • Use proximity ligation assays to detect AKAP10-PKA interactions in situ

• Therapeutic target exploration:

  • Screen for compounds that modulate AKAP10-PKA interactions

  • Test effects on cardiac rhythm in cell and animal models

  • Monitor changes in AKAP10 localization and post-translational modifications