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Phospho-P70S6K (Thr389) Cellular Assay Kit   HTRF® cellular assay kit for measuring endogenous phospho-P70S6K protein directly in cells. Based on our TR-FRET homogeneous and robust HTRF® technology, the phospho-P70S6K assay kit is designed for the detection and analysis of dose-response inhibition and for activation studies of endogenous P70S6K, phosphorylated at Thr389, directly in your cell type of interest. Using a streamlined mix-and-read no-wash protocol, this kit is amenable for any use from basic research to all drug screening phases.   Features Convenient and fast, due to a homogeneous, non-radioactive, one step mix-and-read protocol without any washing Adaptable to all needs from low to ultrahigh throughput High sensitivity, for studying physiologically relevant endogenous levels of P70S6K Reliable and reproducible results, due to robust cryptate chemistry, mean high Z' scores Compatible with most liquid handling systems and TR-FRET multimode plate readers All reagents in one kit, ready to use   Applications Suitable for exploring endogenous translational control signaling Screening and studying of compounds biologically impacting cell proliferation, survival, invasion, motility and insulin receptor signaling Compatible with most cell types Oncology, metabolism (diabetes, obesity, aging), cardiovascular diseases, neurodegenerative diseases      Assay Principle   The phospho-P70S6K assay kit can be run with cell lysates or whole cells.   Upon activation, protein P70S6K is phosphorylated on Thr389. After cell lysis, phospho-P70S6K can be detected and studied using the HTRF® kit reagents. This sandwich immunoassay involves two monoclonal antibodies: an anti-phospho-P70S6K antibody labelled with Eu3+ cryptate and an anti-P70S6K antibody labelled with d2. These antibodies may be pre-mixed and added in a single dispensing step for direct detection.     The assay can be run under a two-plate protocol or be further streamlined to a one-plate assay.  TWO-PLATE ASSAY PROTOCOL   Cells are plated, stimulated and next lysed in the same 96-well culture plate. Lysates are then transferred to the assay plate for the detection of phosphorylated P70S6K by HTRF® reagents. This protocol enables the cells' viability and confluence to be monitored.  ONE-PLATE ASSAY PROTOCOL   Detection of phosphorylated P70S6K with HTRF® reagents is performed in a single plate used for plating, stimulation, lysis and detection. No washing steps are required. This protocol, designed for HTS, enables miniaturization while maintaining a high quality output.         Simplified pathway  P70S6K is a pro-survival factor which belongs to the family of serine/threonine protein kinases. P70S6K acts downstream of the mammalian target of Rapamycin (mTOR), and is crucial for the regulation of cell growth, proliferation, survival and migration by its signaling to several important downstream effectors, e.g. S6RP, elF4B and eEF2K, which induces protein synthesis. P70S6K has multiple functions: it is also involved in insulin receptor signaling by regulating the insulin substrate (IRS1), and has a survival effect by negatively regulating apoptosis via its control of the pro-apoptotic protein, Bad.       Product Performance 1. Western Blot versus HTRF assay Human HEK293 cells were grown in a T175 flask at 37°C, 5% CO2, for 1 day. Day 2: After removal of cell culture medium, 3ml of supplemented lysis buffer were added and incubated for 30 minutes. Soluble supernatants were collected after a 10 minute centrifugation. Equal amounts of lysates were used for a side by side comparison of Western Blot and HTRF®.3000 cells can be detected by using HTRF® phospho-P70S6K (Thr389) whereas 6000 cells are needed for the Western Blot. The HTRF® assay is 2-fold more sensitive than Western Blot.          2. Pharmacological response: agonist and antagonist action modes Human HEK293 cells (100,000 cells/well) were incubated with two concentrations of the inhibitors indicated, Wortmanin and LY294002, followed by stimulation with 1.0 µM of Insulin for 30 minutes at 37°C. After 30 minutes of lysis incubation, phosphorylated P70S6K was measured using the two-plate assay protocol           3. Rapamycin inhibition on stimulated NIH3T3 cells Murine NIH3T3 cells (100,000 / 50,000 / 25,000 cells/well) were incubated for 3 hours with varying concentrations of Rapamycin inhibitor, followed by stimulation with 1.0 µM of Insulin for 30 minutes at 37°C. After 30 minutes of lysis incubation, inhibition of P70S6K phosphorylation was measured using the HTRF® phospho-P70S6K (Thr389) assay with two-plate protocol.          

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