Figure 1: Treatment of NIH/3T3 cells with PDGF stimulates phosphorylation of Akt3 at Ser472, detected by PathScan® Phospho-Akt3 (Ser472) Sandwich ELISA Kit (Mouse Preferred) #7942, but does not affect levels of total Akt3 protein detected by PathScan® Total Akt3 Sandwich ELISA Kit (Mouse Preferred) #7934. The absorbance readings at 450 nm are shown in the top figure while the corresponding western blots using Akt3 (62A8) Rabbit mAb #3788 (left panel) and Phospho-Akt (Ser473) (193H12) Rabbit mAb #4058 (right panel) are shown in the bottom figure.
Figure 2: Demonstration of phospho-Akt sandwich ELISA kit specificity using recombinant human phosphorylated Akt1, Akt2 and Akt3 proteins. Phospho-Akt1 (Ser473) is detected by PathScan® Phospho-Akt1 (Ser473) Sandwich ELISA Kit #7160, Phospho-Akt2 (Ser474) is detected by PathScan® Phospho-Akt2 (Ser474) Sandwich ELISA Kit #7048, while PathScan® Phospho-Akt3 (Ser472) Sandwich ELISA Kit (Mouse Preferred) #7942 measures levels of phospho-Akt3 (Ser472). Recombinant phosphorylated Akt protein (1.0 ng/microwell) is assayed using each ELISA kit.
Figure 3: The relationship between protein concentration of lysates from untreated and PDGF-treated NIH/3T3 cells and the absorbance at 450 nm is shown. After starvation, NIH/3T3 cells (85% confluence) were treated with PDGF #9909 (50 ng/ml) for 10 min at 37ºC and then lysed.
The PathScan® Phospho-Akt3 (Ser472) Sandwich ELISA Kit (Mouse Preferred) is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Akt3 protein phosphorylated at Ser472. A phospho-Akt rabbit mAb has been coated onto the microwells. After incubation with cell lysates, phospho-Akt protein is captured by the coated antibody. Following extensive washing, an Akt3 mouse mAb is added to detect the captured phospho-Akt3 protein. Anti-mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate TMB is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of Akt3 phosphorylated at Ser472.
PathScan® Phospho-Akt3 (Ser472) Sandwich ELISA Kit (Mouse Preferred) detects endogenous levels of Akt3 protein phosphorylated at Ser472 in mouse cells, as shown in Figure 1. Phospho-Akt3 kit specificity is demonstrated in Figure 2, while the kit sensitivity is shown in Figure 3. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Akt, also referred to as PKB or Rac, plays a critical role in controlling survival and apoptosis (1-3). This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin-sensitive pathway involving PI3 kinase (2,3). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (4) and by phosphorylation within the carboxy terminus at Ser473. The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin (mTOR) in a rapamycin-insensitive complex with rictor and Sin1 (5,6). Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets, including Bad (7), forkhead transcription factors (8), c-Raf (9), and caspase-9. PTEN phosphatase is a major negative regulator of the PI3 kinase/Akt signaling pathway (10). LY294002 is a specific PI3 kinase inhibitor (11). Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK-3α and β (12,13). Akt may also play a role in insulin stimulation of glucose transport (12). In addition to its role in survival and glycogen synthesis, Akt is involved in cell cycle regulation by preventing GSK-3β-mediated phosphorylation and degradation of cyclin D1 (14) and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 (15) and p21 Waf1/Cip1 (16). Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin-sensitive complex containing raptor (17). More importantly, Akt phosphorylates and inactivates tuberin (TSC2), an inhibitor of mTOR within the mTOR-raptor complex (18,19).
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