Reaction buffer

Recombinant GST-PAH (vector synthesized by Vectorbuilder) was produced in Escherichia coli (BL21) as GST fusion protein, and purified by affinity chromatography on glutathione–Sepharose columns. Recombinant human proteins, PKD3 and PKA, were both purchased from Enzo biosciences and SignalChem Biotech, respectively. Kinase reactions were performed in reaction buffer (Cell Signaling Technology) in the presence of cold (nonradioactive) ATP (Cell Signaling Technology) for 30 min at 30°C. As indicated in the experiment, 1 μM of CRT0066101 (Tocris) was added to the corresponding condition. Proteins from the kinase reactions were boiled in 5× Laemmli buffer and analyzed by Western blotting. Membrane was incubated with the appropriate primary antibody against the phosphorylated motif (RxxS/T*) (Cell Signaling Technology), PAH (proteintech, PK), and PKD3 (Cell signaling Technology).

To determine mTOR kinase activity, an ATP assay was carried out using the ADP-Glo Kinase Assay Kit, in accordance with the manufacturer’s instructions (Promega, Madison, WI, USA). The active recombinant mTOR (50 ng) protein was mixed with different concentrations of 2,6-DMBQ, AZD8055 (dissolved in DMSO) as a mTOR inhibitor, or vehicle (DMSO) in reaction buffer (Cell Signaling Technology) and incubated at room temperature for 15 min. The inactive p70S6K recombinant protein (100 ng) and ATP were added and the mixtures were incubated at 30 °C for 30 min. The fluorescence of each sample was measured at excitation and emission wavelengths of 530 nm and 590 nm, respectively.