The purified mTOR protein (Cloud-Clone Corp.,Wuhan, China) was used as the substrate for an in vitro kinase assay with 100 ng of active AKT1 (Signalchem, Catalog No. A16-10G) or p70S6K (Signalchem, Catalog No. R21-10H). Reactions were conducted in 1X kinase buffer (25 mM Tris-HCl pH 7.5, 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2, and 5 mM MnCl2) containing 200 μM ATP at 30°C for 30 min. Reactions were stopped by adding 6X protein loading buffer, and proteins were detected using western blot analysis.
P70s6k
Manufactured by Sino Biological
Sourced in China
P70S6K is a mammalian serine/threonine protein kinase that plays a key role in the regulation of cell growth, proliferation, and survival. It is a downstream effector of the PI3K/Akt signaling pathway and is involved in the control of protein synthesis and cell cycle progression.
Automatically generated - may contain errors
2 protocols using p70s6k
1
In Vitro Kinase Assay of mTOR
2
Phosphorylation of PHD2 by P70S6K
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The purified wild-type or mutant PHD2-FLAGM2 proteins (100 ng) were incubated in kinase buffer (25 mM 3-Morpholinopropane-1-sulfonic acid (MOPS) [pH 7.2], 25 mM MgCl2, 5 mM EGTA, 2 mM EDTA, 250 μM DTT, and 6 mM β-glycerophosphate) in the presence of active P70S6K (4 ng/μL, SignalChem), 250 μM ATP, and 2 μCi γ-32P-ATP (PerkinElmer). After 20-min incubation at 30°C, samples were denaturated by incubating with 4× SDS sample buffer at 95°C for 10 min and separated by 10% SDS-PAGE. Thereafter, the gel was blotted onto a nitrocellulose membrane, and the membrane was exposed to a Kodak phosphor imaging screen overnight. Autoradiography was detected with MolecularImager FX using Quantity One software (all from Bio-Rad). Afterward, the total proteins were detected by using an antibody against PHD2.
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