Supersignal west femto maximum sensitivity substrate ecl kit

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.