In vitro kinase assay

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).