Phospho akt thr450

DSS salt (colitis grade, molecular weight: 36,000–50,000 Daltons) was purchased from MP Biomedicals (Ohio, USA). The hematoxylin-eosin (H&E) staining solution was purchased from Sigma (St. Louis, MO, USA). Trizol reagent was obtained from Invitrogen (Carlsbad, USA), and Primescript™ RT reagent kits and SYBR^® Premix Ex Taq™ II kits were acquired from Takara Bio Inc. (Kusatsu, Japan). Antibodies including p44/42 MAPK (Erk1/2), phospho-p44/42 MAPK (Thr202/Tyr204), p38 MAPK, phospho-p38 MAPK (Thr180/Tyr182), SAPK/JNK, phospho-SAPK/JNK (Thr183/Tyr185), Akt, phospho-Akt (Thr450), and beta-Actin were purchased from Cell Signaling Technology (MA, USA).

mTORC2 kinase activity assays were conducted in 100 mM Hepes (pH 7.4), 1 mM EGTA, 1 mM TCEP, 0.0025% Tween 20, and 10 mM MnCl₂ using dephosphorylated Akt1 as a substrate. In a 60-μl reaction volume, 0.05 μM of either WT or A- and I-site mutant mTORC2 was mixed with 1 μM Akt1 and, where indicated, either dimethyl sulfoxide or 25 μM Torin1. The mixture was preincubated for 5 min at room temperature, and the reaction was initiated by the addition of 10 μM ATP. After 20 min at 37°C, the reaction was terminated by the addition of 60 μl of 2× Laemmli sample buffer. The reactions were analyzed by Western blotting using primary antibodies against phospho–AKT-Ser⁴⁷³ (no. 4060; Cell Signaling Technology, Beverly, USA), phospho–AKT-Thr⁴⁵⁰ (no. 9267; Cell Signaling Technology, Beverly, USA), AKT (no. 4685), and mTOR (no. 2972; Cell Signaling Technology, Beverly, USA), anti-FLAG antibodies (Sigma-Aldrich, F1804), SIN1 (Bethyl, A300-910A), and Rictor (Bethyl, A300-458A) at a dilution of 1:1000 in 5 ml of TBST. A goat anti-rabbit HRP-labeled antibody (ab6721; Abcam, Cambridge, UK) was used as the secondary antibody at a dilution of 1:3000 in 5 ml of TBST. Signals were detected using the Enhanced Chemiluminescence (ECL) Kit SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). Images were acquired using a Fusion FX (Vilber) imaging system.