PRKAR2B (Ab-113) Antibody

What is PRKAR2B and what cellular functions does it regulate?

PRKAR2B, also known as PKA R2-beta or KAP3, functions as a regulatory subunit of cAMP-dependent protein kinase (PKA). It plays a critical role in modulating PKA activity, which is central to numerous cellular processes. PRKAR2B is particularly important in adipocyte differentiation, where it serves as a positive regulator of adipogenesis. Studies have demonstrated that PRKAR2B expression progressively increases during adipocyte differentiation, suggesting its crucial role in this developmental process . Additionally, PRKAR2B has been implicated in cancer progression, particularly in prostate cancer where it contributes to proliferation and metastasis .

How does PRKAR2B interact with the PKA signaling pathway?

PRKAR2B regulates PKA activity by binding to catalytic subunits in the absence of cAMP. When cAMP levels increase, the regulatory subunits bind cAMP, causing conformational changes that release the catalytic subunits, thereby activating PKA. Interestingly, recent research has revealed that PRKAR2B can regulate PKA activity through mechanisms independent of cAMP flux. For example, in Theileria annulata-transformed macrophages, miR-34c-3p-mediated ablation of PRKAR2B represents an epigenetic mechanism for regulating mammalian PKA activity independent of fluctuations in cAMP levels .

What are the technical specifications of PRKAR2B (Ab-113) antibody?

The PRKAR2B (Ab-113) antibody is a polyclonal antibody derived from rabbit that specifically recognizes human PKA-R2β around the phosphorylation site of serine 113 (R-A-S(p)-V-C). The antibody has the following characteristics:

The antibody is specifically designed to detect endogenous levels of total PKA-R2 beta protein .

How should PRKAR2B (Ab-113) antibody be stored for optimal performance?

The PRKAR2B (Ab-113) antibody should be stored at -20°C or -80°C upon receipt. It is supplied in a buffer containing 50% glycerol, which helps maintain stability during freezing. Repeated freeze-thaw cycles should be avoided as they can compromise antibody performance. For short-term storage (less than one week), the antibody can be kept at 4°C. When handling the antibody, it is advisable to aliquot it into smaller volumes to minimize the number of freeze-thaw cycles .

What are the validated applications for PRKAR2B (Ab-113) antibody?

The PRKAR2B (Ab-113) antibody has been validated for several experimental techniques:

• Western Blotting (WB): Effective for detecting PRKAR2B protein expression levels in cell and tissue lysates.

• Enzyme-Linked Immunosorbent Assay (ELISA): Suitable for quantitative measurement of PRKAR2B in solution.

• Immunohistochemistry (IHC): Validated for detecting PRKAR2B in fixed tissue sections.

• Immunofluorescence (IF): Can be used to visualize the subcellular localization of PRKAR2B.

• Immunocytochemistry (ICC): Effective for detecting PRKAR2B in cultured cells .

When using this antibody for Western blotting, researchers should expect to detect a band at approximately 52 kDa, which corresponds to the molecular weight of PRKAR2B.

How should Western blotting protocols be optimized for PRKAR2B detection?

For optimal detection of PRKAR2B in Western blotting applications, consider the following methodological recommendations:

• Sample Preparation: Use RIPA buffer supplemented with protease and phosphatase inhibitors for efficient protein extraction.

• Gel Concentration: A 10-12% SDS-PAGE gel is recommended for optimal separation of PRKAR2B (approximately 52 kDa).

• Transfer Conditions: Transfer proteins to PVDF membrane at 100V for 60-90 minutes in cold transfer buffer containing 20% methanol.

• Blocking: Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary Antibody Incubation: Dilute PRKAR2B (Ab-113) antibody at 1:500 to 1:2000 in blocking buffer and incubate overnight at 4°C.

• Detection: Use appropriate HRP-conjugated secondary antibody and develop using enhanced chemiluminescence.

For detecting phosphorylated forms of PRKAR2B, BSA is preferred over milk for blocking and antibody dilution, as milk contains phosphoproteins that may interfere with detection .

What role does PRKAR2B play in adipogenesis?

PRKAR2B has been identified as the main regulatory subunit of PKA in both murine and human adipose tissue during development. Research has demonstrated that PRKAR2B expression progressively increases during adipocyte differentiation, suggesting its crucial involvement in adipogenesis. Functional studies using siRNA-mediated silencing have provided compelling evidence for PRKAR2B's essential role in this process:

• Reduced Lipid Accumulation: Silencing of PRKAR2B in both 3T3-L1 cells and primary human preadipocytes resulted in significantly reduced triglyceride accumulation compared to control cells.

• Impaired Expression of Adipogenic Markers: PRKAR2B silencing abolished the normal increase in expression of key adipocyte differentiation markers including PPARγ, FABP4, FAS, and LPL that typically occurs during differentiation.

• Compensatory Mechanisms: When PRKAR2B is silenced, a compensatory increase in PRKAR1A expression occurs, suggesting a complex interplay between PKA regulatory subunits .

These findings collectively establish PRKAR2B as a positive regulator of adipogenesis that is required for both murine and human adipocyte differentiation.

How does PRKAR2B silencing affect the molecular cascades in adipocyte differentiation?

Silencing PRKAR2B disrupts several key molecular events in adipocyte differentiation:

• Transcription Factor Expression: PRKAR2B silencing prevents the normal increase in PPARγ expression, which is a master regulator of adipogenesis. Without adequate PPARγ, the entire adipogenic program is compromised.

• Lipid Metabolism Genes: The expression of genes involved in lipid metabolism and storage, such as Fatty Acid Binding Protein 4 (FABP4), Fatty Acid Synthase (FAS), and Lipoprotein Lipase (LPL), is significantly impaired in PRKAR2B-silenced cells.

• Mitotic Clonal Expansion (MCE): Though MCE occurs normally in PRKAR2B-silenced 3T3-L1 cells, the subsequent gene expression changes required for differentiation are blocked, indicating that PRKAR2B acts downstream of the initial proliferative response in adipogenesis .

These findings suggest that PRKAR2B is a crucial component in the signaling network that regulates adipocyte differentiation, potentially by modulating the activity of PKA, which in turn affects the expression and activity of key adipogenic transcription factors.

How do microRNAs regulate PRKAR2B expression?

PRKAR2B expression is regulated by several microRNAs that play crucial roles in different biological contexts:

• miR-34c-3p Regulation: Research has identified PRKAR2B as a direct target of miR-34c-3p. In the context of Theileria annulata parasite infection, miR-34c-3p targets PRKAR2B, thereby modulating PKA activity and influencing the dissemination phenotype of Theileria-transformed macrophages. Computational analysis using miRWalk and TargetScan algorithms confirmed PRKAR2B as a potential target of miR-34c-3p in the Bos taurus genome .

• miR-200 Family Regulation: PRKAR2B has also been identified as a target of the miR-200 family in the central nervous system (CNS). Experimental evidence from luciferase reporter assays demonstrated that overexpression of miR200b/a/429 together with a plasmid carrying the wild-type 3′UTR of PRKAR2B reduced luciferase signal, confirming direct targeting. When the seed sequences in the 3′UTR of PRKAR2B were inverted or replaced, this effect was abolished, further validating PRKAR2B as a direct target of miR200 family members .

These microRNA-mediated regulatory mechanisms represent an important layer of post-transcriptional control over PRKAR2B expression, with significant implications for various physiological and pathological processes.

What are the functional consequences of miR-34c-3p-mediated PRKAR2B regulation?

The miR-34c-3p-mediated regulation of PRKAR2B has several significant functional consequences:

• PKA Activity Modulation: By targeting PRKAR2B, miR-34c-3p affects PKA activity independent of changes in cAMP levels, representing an epigenetic mechanism for regulating mammalian PKA signaling.

• Transformed Macrophage Phenotype: In Theileria annulata-transformed macrophages, reduced PRKAR2B expression through miR-34c-3p action influences the dissemination phenotype of these cells, potentially affecting disease progression.

• Cancer and Metabolic Implications: Beyond parasitic infections, this regulatory mechanism may have implications for certain human cancers where PRKAR2B is highly expressed. For instance, in prostate cancer, PRKAR2B is involved in proliferation and metastasis, suggesting that miR-34c-3p mimics could potentially have therapeutic applications .

• Metabolic Regulation: There is preliminary evidence suggesting that miR-34c-3p regulation of PRKAR2B might also be relevant in brown adipocytes, pointing to potential roles in metabolism and possibly obesity treatment .

The discovery of these regulatory relationships offers new perspectives for understanding disease mechanisms and developing potential therapeutic approaches targeting the miR-34c-3p/PRKAR2B axis.

What are common issues when using PRKAR2B antibodies and how can they be resolved?

When working with PRKAR2B antibodies, researchers may encounter several technical challenges:

• Non-specific Binding:

  • Problem: Multiple bands appearing in Western blot.

  • Solution: Increase blocking time (2-3 hours), use 5% BSA instead of milk, optimize antibody dilution (try 1:1000-1:2000), and include additional washing steps.

• Weak Signal:

  • Problem: Faint or no detectable bands.

  • Solution: Increase protein loading (50-80 μg), reduce antibody dilution, extend primary antibody incubation (overnight at 4°C), and ensure fresh detection reagents.

• Background Issues in Immunostaining:

  • Problem: High background in immunofluorescence or immunohistochemistry.

  • Solution: Extend blocking time, use more stringent washing, optimize antibody concentration, and consider using a different secondary antibody.

• Cross-reactivity with Other Proteins:

  • Problem: Detecting non-target proteins.

  • Solution: Validate antibody specificity using PRKAR2B-knockout/knockdown controls and pre-absorption with the immunizing peptide.

• Inconsistent Results Between Experiments:

  • Problem: Variable results across different experiments.

  • Solution: Standardize protein extraction methods, create a detailed protocol with consistent timing, and include appropriate positive and negative controls in each experiment.

How can researchers validate the specificity of PRKAR2B (Ab-113) antibody?

Validating antibody specificity is crucial for generating reliable experimental data. For PRKAR2B (Ab-113) antibody, consider these validation approaches:

• Genetic Validation:

  • Perform siRNA knockdown of PRKAR2B and confirm reduced signal in Western blot, immunofluorescence, or other applications.

  • Use CRISPR-Cas9 to generate PRKAR2B knockout cells as a negative control.

• Peptide Competition Assay:

  • Pre-incubate the antibody with the immunizing peptide (the synthesized non-phosphopeptide derived from human PKA-R2β around serine 113) before application.

  • A specific antibody will show diminished or absent signal after peptide competition.

• Multiple Antibody Validation:

  • Compare results using different PRKAR2B antibodies targeting distinct epitopes.

  • Consistent results across different antibodies increase confidence in specificity.

• Correlation with mRNA Expression:

  • Compare protein detection with PRKAR2B mRNA levels measured by qPCR.

  • Protein and mRNA levels should show similar patterns in response to experimental manipulations.

• Subcellular Localization Confirmation:

  • Verify that the detected protein localizes to cellular compartments consistent with known PRKAR2B distribution.

Following these validation strategies will ensure the reliability and reproducibility of results obtained using the PRKAR2B (Ab-113) antibody .

What is the significance of PRKAR2B in cancer development and progression?

PRKAR2B exhibits important roles in several cancer types, with particularly strong evidence in prostate cancer:

• Prostate Cancer: PRKAR2B is highly expressed in prostate cancer, where it contributes to both proliferation and metastasis. The elevated expression of PRKAR2B in prostate tumor tissues suggests its potential as a biomarker or therapeutic target .

• Signaling Pathway Involvement: As a regulatory subunit of PKA, PRKAR2B affects various signaling pathways that control cell cycle progression, apoptosis resistance, and invasive capability - all hallmarks of cancer progression.

• microRNA Regulation: The regulation of PRKAR2B by microRNAs such as miR-34c-3p may be dysregulated in cancer cells, suggesting that microRNA mimics or antagonists targeting this regulatory mechanism could have therapeutic potential in cancer treatment .

Understanding the precise role of PRKAR2B in specific cancer types is still evolving, but the current evidence suggests it may serve as both a biomarker and potential therapeutic target in certain malignancies.

How does PRKAR2B function in parasitic disease models like Theileria annulata infection?

In Theileria annulata infection models, PRKAR2B plays a significant role in host-parasite interactions:

• Transformation Mechanism: Theileria annulata is a unique intracellular parasite that transforms bovine leukocytes. This transformation process involves manipulation of host cell signaling pathways, including PKA activity.

• Three-Pronged Regulation of PKA: T. annulata infection and transformation of bovine macrophages targets mammalian PKA activity through three distinct mechanisms:

  • Raising cAMP levels

  • Suppressing the endogenous PKA inhibitor PKIG

  • Facilitating miR-34c-3p-mediated ablation of PRKAR2B expression

• Impact on Cell Dissemination: These alterations in PKA signaling, particularly through PRKAR2B regulation, influence the dissemination potential of Theileria-transformed macrophages, which is crucial for parasite spread and disease progression .

• Therapeutic Implications: The identification of miR-34c-3p as a regulator of PRKAR2B in this context suggests that microRNA mimics or antagonists could potentially be developed as therapeutic agents for treating tropical theileriosis, a significant animal disease affecting cattle in many regions .

This research highlights the sophisticated mechanisms by which parasites can manipulate host cell signaling through targeting regulatory components like PRKAR2B, and suggests novel approaches for intervention.

How can PRKAR2B antibodies be used in phosphorylation studies?

The PRKAR2B (Ab-113) antibody is particularly valuable for phosphorylation studies because it recognizes the region around the phosphorylation site of serine 113 (R-A-S(p)-V-C). This makes it useful for several advanced research applications:

• Phosphorylation State Analysis: The antibody can be used to monitor changes in PRKAR2B phosphorylation status under different cellular conditions, such as before and after treatment with agents that affect cAMP levels or PKA activity.

• Kinase/Phosphatase Assays: Researchers can employ this antibody to assess the activity of kinases or phosphatases that might target this specific residue by monitoring changes in phosphorylation levels.

• Signaling Pathway Dynamics: By tracking PRKAR2B phosphorylation alongside other signaling molecules, researchers can map the temporal dynamics of signaling cascades involving PKA.

• Methodological Considerations:

  • For phosphorylation studies, samples should be prepared with phosphatase inhibitors to preserve phosphorylation status.

  • Using BSA rather than milk for blocking is essential, as milk contains phosphoproteins that can interfere with phospho-specific detection.

  • Parallel detection with phospho-specific and total PRKAR2B antibodies provides the most informative results .

What experimental approaches are recommended for studying PRKAR2B-microRNA interactions?

To investigate PRKAR2B regulation by microRNAs such as miR-34c-3p or miR-200 family members, researchers can employ these methodological approaches:

• Luciferase Reporter Assays:

  • Clone the PRKAR2B 3'UTR downstream of a luciferase reporter gene.

  • Create mutant versions with altered microRNA binding sites.

  • Co-transfect cells with the reporter constructs and microRNA mimics or inhibitors.

  • A decrease in luciferase activity with wild-type 3'UTR but not with mutated binding sites confirms direct targeting, as demonstrated with miR-200 family members .

• Expression Analysis:

  • Transfect cells with microRNA mimics or inhibitors and measure changes in PRKAR2B mRNA (by qPCR) and protein (by Western blot) levels.

  • Use correlation analyses to assess relationships between endogenous microRNA and PRKAR2B expression levels across different cell types or conditions.

• Functional Rescue Experiments:

  • Perform knockdown of PRKAR2B using siRNA and assess the resulting phenotype.

  • Attempt to rescue the phenotype by inhibiting the targeting microRNA.

  • Alternatively, induce phenotypes by overexpressing the microRNA and attempt rescue by expressing a PRKAR2B construct lacking the microRNA binding sites.

• In Vivo Verification:

  • Use transgenic models with altered expression of the microRNA of interest.

  • Compare PRKAR2B levels in tissues from wild-type and transgenic animals, as demonstrated in Foxg1cre/+ hippocampus studies .

These approaches can provide comprehensive insights into the regulatory relationship between PRKAR2B and its targeting microRNAs in various biological contexts.