HTRF assay

The HTRF assay was performed as reported previously^{68, 69} with minor adjustments. Briefly, substrates of JNK-1 and Erk-1 (Life, Carlsbad, CA, USA) were mixed and incubated with corresponding kinases to explore the optimal enzyme conditions with varying reaction times and concentrations. The criteria of enzyme optimization were (1) the highest slope indicating the widest reaction window and the most sensitive reaction condition to guarantee that the end point of an individual reaction was within the linear kinetic curve and (2) the restricted amount of enzymes. Then, the apparent ANP K_m for each kinase was determined⁷⁰ (K_mis the Michaelis constant for ANP) (excitation wavelength: 320 nm; emission wavelength: 620 nm). A volume of 2.5 μl of drugs, containing 10 μM PL, 50 μM ZA or 10 μM PL plus 50 μM ZA at equal volumes, was mixed and supplemented with 2.55 μl ATP/well (Sigma, St. Louis, MO, USA) and 5 μl/well of substrate and kinase mixture to start the enzymatic steps. This reaction was carried out at room temperature for 1 h. The 10 μl antibody mixtures containing 5 ng/μl anti-GST-XL665 (Cisbio China, Shanghai, China) and 0.0225 ng/μl anti-pATF2-EuK (Cisbio China, Shanghai, China) were used at room temperature for 1 h to stop the enzymatic reaction. The OD of drugs' IC₅₀ at 665 nm/620 nm was detected with a multimode microplate reader (Tecan Trading AG, Mannedorf, Zurich, Switzerland) (a drug's IC₅₀ value signifies the potency of inhibition based on the concentration of inhibitor where 50% inhibition is reached).