Akt Signaling Pathway Antibodies

 

Akt is one of the most actively studied kinase pathways in the basic research and drug development arenas, as it plays an integral role in mediating signals for cell growth, survival, cell-cycle progression, differentiation, transcription, translation, and glucose metabolism. Recent advances in Akt signaling have focused on understanding cellular processes and identifying cellular substrates that are physiologically relevant in vivo. These efforts have uncovered important roles for Akt pathway regulation in cancer research, neuroscience, and disease prevention.

There are three highly homologous members known as Akt1 or simple Akt, Akt2, and Akt3 with alternatively named PKB α, PKB β, and PKB γ, respectively. Akt1 plays important role in cellular survival by inhibiting apoptotic processes and implicated as a major factor in many types of cancer. Akt2 required to induce glucose transport is an important signaling molecule in the Insulin signaling pathway. The role of Akt3 is less clear, though it appears to be predominantly expressed in the brain. Deregulations in the Akt-related pathway were observed in many human diseases, including cancer, cardiopathies, neurological disorders, and type-2 diabetes.

Thanks to the funding support from the National Cancer Institute (NCI Awards HHSN26100900070C and HHSN261201100087C), Rockland has developed over 100 Akt-related products including a suite of reagents, and antibody-based assays. Additionally, recombinant AKT calibrator proteins have been produced as both Akt active (phosphorylated) and non-active (phosphatase-treated and phosphorylation site double mutant recombinant proteins). These reagents can be used for detecting status of proteins involved in PI3K/AKT/mTOR pathway including Akt1, Akt2 and Akt3 isoforms and their phosphorylated stages.



Profiling AKT Protein Activation Status

Akt is activated by phosphorylation at the Ser473 and Thr308 positions by mTORC2 and PDK1 kinases, respectively. Faulty activation of Akt underlies the pathophysiological properties of a variety of complex diseases, including type-2 diabetes and cancer. Constitutively activated, Akt promotes cellular survival as well as resistance to treatment with chemotherapy and radiation therapy. In fact, several research groups are developing compounds targeting Akt as anticancer drugs. Quantitation of the expression of Akt proteins and their activated status in response to the pathway inhibitors would have great potential application in monitoring therapeutic efficacy.

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Figure 1: Localization of AKT (Green ) by fluorescence microscopy using RABBIT Anti-AKT Antibody

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Figure 2: Localization of active AKT (red) in human brain cerebellum tissue using Mouse Anti-AKT pT308 Antibody

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Figure 3: Western blot using A431 cells stimulated with EGF and Mouse Anti-AKTpS473 antibody



Pharmacodynamic assay for AKT/mTOR inhibitors

AKT/PI3K/mTOR pathway become a major focus of the research community thanks to its pivotal regulatory role in diverse cellular processes, including cell survival, cancer progression and insulin metabolism. The Akt cascade is activated by a number of stimuli such as receptor tyrosine kinases, G-protein-coupled receptors, cytokine receptors integrins, and B and T cell receptors. In recent years, alongside the well-characterized Akt1 isoform, Akt2 and Akt3 have emerged as significant contributors to cancer with distinct non-overlapping functions. The Akt pathway is attractive target for developing anti-cancer drugs as some experimental cancer drugs (Akt inhibitors e.g., Perifosine; mTOR inhibitors e.g., rapamycin; PI3K inhibitors e.g., LY294002) have been developed and used in clinical trials.

Isoforms of AKT are found to be distinct with regard to tissue expression, pathway activation and inhibitor sensitivity. Rockland has created a panel of monoclonal antibodies to AKT isoforms. These anti-AKT antibodies are available to academic researchers as well as biopharma companies who wish to manufacture highly sensitive pharmacodynamic assays for measuring both total AKT, its isoforms and levels of phosphorylated Akt in response to mTOR / AKT inhibitors and modulators.

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Figure 4: Immunofluorescence Microscopy of Mouse Anti-AKTpS473 antibody using STED nanoscopy to evaluate AKT activation and migration. Panel A: serum starved, unstimulated A431 cells. Panel B: serum starved A431 cells with 15 minute EGF stimulation.

Quantitation of the expression of Akt proteins and their activated status as well as activation and expression status of other proteins involved in Akt pathway in response to the pathway inhibitors would have great potential application in monitoring therapeutic efficacy.

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Figure 5: Dot blot illustrating reactivity of anti-AKT pT308 monoclonal antibody

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Figure 6: Dot blot demonstrating the phosphorylation status of AKT. Recombinant AKT was untreated or treated with phosphatase to remove the phosphorylation at the T308 and S473 sites. Only the phosphorylated AKT is detected by anti AKT pT308.

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Figure 7: Sandwich ELISA assay to profiling active AKT2 Isoform

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Figure 8: Recombinant AKT3 detected with AKT specific antibodies. AKT3 co-expressed with P110 Kinase leads to phosphorylation as demonstrated by reactivity with anti pS473 and pT308 monoclonal antibodies.



AKT Antibodies & Assays for Cell Signaling

Activation of AKT results in phosphorylation of a wide range of proteins downstream of the AKT pathway, making AKT Western blotting a popular technique for monitoring this reaction. The phosphorylated substrates, present in various subcellular locations play an important role in directing different phenotypic outcomes. Faulty or aberrant activation of AKT underlies the pathophysiological properties of a variety of complex diseases, including type-2 diabetes, HIV, and cancer. When constitutively active, AKT is utilized frequently by cancer cells to circumvent therapeutic intervention, promoting cellular survival and resistance to chemo and radiation therapy. Our anti-Akt antibodies and PI3Kinase Pathway reagents help monitor levels of AKT and its phosphorylation at position S473, T308, as well as AKT antibody isoforms AKT2 and AKT3.



Immunoassays for AKT Signaling

There are a variety of immunoassays that can assist in monitoring AKT antibody levels, many of which are offered by Rockland. We have the ability to develop custom assays based on specific applications. The development of a customized immunoassay is initiated by user-defined specifications pertaining to overall research-related design goals and is a process characteristically divided into 4 phases.

Additionally, Rockland has partnered with other leading researchers to develop highly specialized Western blot assays that simultaneously detect several human serum proteins and resting AKT1 and activated phosphorylated AKT1. With over 20 years of experience in the development and validation of multiple assay formats, Rockland demonstrates consistent success in delivering customer solutions and possesses the expertise in optimizing every aspect of an immunoassay.