| Products/Services Used | Details | Operation |
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| 5X Sample Buffer | Cells were solubilized at 4°C for 30 min in RIPA lysis buffer (50 mM Tris-HCl, pH 8.0, 150?mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) with a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, 78442) and then pelleted at 13,400 g for 15?min at 4°C. The supernatant was mixed with 5× sample buffer (GenScript, MB01015), heated to 100°C for 10 min, and then fractionated by SDS–PAGE and transferred to nitrocellulose membranes (Pall, 66485). | Get A Quote |
The class I phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway is a key regulator of cell survival, growth, and proliferation and is among the most frequently mutated pathways in cancer. However, where and how PI3K-AKT signaling is spatially activated and organized in mammalian cells remains poorly understood. Here, we identify focal adhesions (FAs) as subcellular signaling hubs organizing the activation of PI3K-PI(3,4,5)P3-AKT signaling in human cancer cells containing p110α mutations under basal conditions. We find that class IA PI3Ks are preferentially recruited to FAs for activation, resulting in localized production of PI(3,4,5)P3 around FAs. As the effector protein of PI(3,4,5)P3, AKT1 molecules ... More
