Figure 1: Treatment of A-673 cells with insulin stimulates phosphorylation of Akt2 at Ser474, detected by PathScan ® Phospho-Akt2 (Ser474) Sandwich ELISA Kit #7048, but does not affect levels of total Akt2 protein detected by PathScan ® Total Akt2 Sandwich ELISA Kit #7046. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using Akt2 (5B5) Rabbit mAb #2964 (left panel) and Phospho-Akt (Ser473) (D9E) XP ® Rabbit mAb #4060 (right panel) are shown in the bottom figure.
Figure 2: Demonstration of phospho-Akt2 (Ser474) sandwich ELISA kit specificity using recombinant phosphorylated Akt1, Akt2 and Akt3 proteins. Phospho-Akt1 (Ser473) is detected by PathScan ® Phospho-Akt1 (Ser473) Sandwich ELISA Kit #7160 while PathScan ® Phospho-Akt2 (Ser474) Sandwich ELISA Kit #7048 measures levels of phospho-Akt2 (Ser474). Recombinant phosphorylated Akt protein (1.0 ng per microwell) is assayed using both ELISA kits.
Figure 3: The relationship between protein concentration of lysates from untreated and insulin-treated A-673 cells and the absorbance at 450 nm is shown. After starvation, A-673 cells (75% confluence) were treated with insulin (100 nM) for 10-20 min at 37ºC and then lysed.