Figure 1. Target map of the PathScan® Akt Signaling Antibody Array Kit (Chemiluminescent Readout) #9474.
Figure 2. MCF7 cells were grown to 85% confluency and then serum starved overnight. Cells were either untreated (left panel) or treated with Human Insulin-like Growth Factor I (hIGF-I) #8917 (100 ng/ml, 20 min; right panel). Cell extracts were prepared and analyzed using the PathScan® Akt Signaling Antibody Array Kit (Chemiluminescent Readout) #9474. Images were acquired by briefly exposing the slide to standard chemiluminescent film.
Figure 3. A-431 cells were grown to 85% confluency and then serum starved overnight. Cells were either untreated (left panel) or treated with Human Epidermal Growth Factor (hEGF) #8916 (100 ng/ml, 5 min; right panel). Cell extracts were prepared and analyzed using the PathScan® Akt Signaling Antibody Array Kit (Chemiluminescent Readout) #9474. Images were acquired by briefly exposing the slide to standard chemiluminescent film.
The PathScan® Akt Signaling Antibody Array Kit (Chemiluminescent Readout) uses glass slides as the planar surface and is based upon the sandwich immunoassay principle. The array kit allows for the simultaneous detection of 16 phosphorylated proteins predominantly belonging to the Akt signaling network. Target-specific capture antibodies have been spotted in duplicate onto nitrocellulose-coated glass slides. Each kit contains two 16-pad slides, allowing the user to test up to 32 samples and generate 512 data points in a single experiment. Cell lysate is incubated on the slide followed by a biotinylated detection antibody cocktail. Streptavidin-conjugated HRP and LumiGLO® Reagent are then used to visualize the bound detection antibody by chemiluminescence. An image of the slide can be captured with either a digital imaging system or standard chemiluminescent film. The image can be analyzed visually or the spot intensities quantified using array analysis software.
Kit should be stored at 4°C with the exception of Lysis Buffer, which is stored at –20°C (packaged separately).
PathScan® Akt Signaling Antibody Array Kit (Chemiluminescent Readout) detects the indicated cellular proteins and signaling nodes only when phosphorylated at the specified residues (see Array Target Map). No substantial cross-reactivity has been observed between targets. This kit is optimized for cell lysates diluted to a total protein concentration between 0.2 and 1 mg/ml (see kit protocol).
The Akt signaling module is typically activated in response to growth factor stimulation of receptor tyrosine kinases transmitting primarily anabolic growth and survival signals. Akt1/2 are ubiquitously expressed protein kinases having a multitude of cellular substrates and are involved in the regulation of a wide range of cellular processes. Akt is activated by phosphorylation at two distinct sites: Ser473 by the mTORC2 complex and Thr308 by the plasma membrane residing kinase PDK1.
PI3 kinase is a lipid kinase that phosphorylates inositol phospholipids at position three to generate docking sites for Akt at the plasma membrane where Akt is activated. PTEN is a lipid phosphatase that negates the action of PI3 kinase to downregulate the signal emanating from this module.
mTOR integrates growth factor signaling and nutrient availability and is a core component of two macromolecular complexes, mTORC1 and mTORC2. The autophosphorylation of mTOR at Ser2481 correlates with the levels of its activation. mTORC1 phosphorylation of p70 S6 kinase leads to kinase activation, which in turn activates protein synthesis. The S6 ribosomal protein is found downstream of p70 S6 kinase and its phoshporylation at Ser235/236 reflects mTOR pathway activation. The mTORC2 complex activates Akt by phosphorylating it at Ser473. Phosphorylation of PRAS40 at Thr246 by Akt relieves PRAS40 inhibition of mTORC1.
4E-BP1 is a repressor of translation and inhibits cap-dependent translation initiation. Hyperphosphorylation of 4E-BP1 by mTORC1 leads to derepression of this blockade, which results in activation of cap-dependent translation.
Phosphorylation of the pro-apoptotic protein Bad at Ser112 and the multifunctional kinases GSK-3α and GSK-3β at Ser21 and Ser9, respectively, by Akt inhibits their activity and promotes cell survival.
AMPK is an energy sensor that is activated by phosphorylation at Thr172 in response to elevated AMP levels. Under conditions of low energy and elevated levels of AMP, AMPK helps to ensure that anabolic processes, such as those triggered by Akt, are decreased until energy levels are restored.
Although not a component of the Akt signaling network, Erk1 and Erk2 kinases are a central component of the Ras/MAP kinase signaling module. Erk1/2 regulate multiple cellular functions and are involved in a broad range of cellular processes, such as proliferation, differentiation, and motility. Erk and Akt signaling modules cross regulate each other at multiple points and through a variety of mechanisms. Erk is activated by a wide range of extracellular signals including growth factors, cytokines, hormones, and neurotransmitters, leading to dual phosphorylation at Thr202 and Tyr204.
The 90 kDa ribosomal S6 kinase 1 (RSK1) is activated primarily by Erk1/2 in response to many growth factors, polypeptide hormones, and neurotransmitters. p90RSK1 phosphorylates a wide range of substrates including ribosomal protein S6, and positively regulates protein translation and cellular growth. p90RSK1 can also be activated by kinases that regulate the response to cellular stress