4.8. Cellular Thermal Shift Assay (CETSA)

Engagement between each compound and tNOX in cells was analyzed by CETSA. Samples were prepared from control and drug-exposed cells. For each set, 2 × 10⁷ cells were seeded in a 10-cm cultured dish. After 24 h of culture, the cells were pretreated with 10 μM MG132 for 1 h, washed with PBS, treated with trypsin, and collected. Samples were centrifuged at 12,000 rpm for 2 min at room temperature, the pellets were gently resuspended with 1 mL of PBS, and the samples were centrifuged at 7500 rpm for 3 min at room temperature. The pellets were resuspended with 1 mL of PBS containing 20 mM Tris-HCl pH 7.4, 100 mM NaCl, 5 mM EDTA, 2 mM phenylmethylsulfonyl fluoride (PMSF), 10 ng/mL leupeptin, and 10 μg/mL aprotinin. The samples were transferred to Eppendorf tubes and subjected to three freeze-thaw cycles; for each cycle, they were exposed to liquid nitrogen for 3 min, placed in a heating block at 25 °C for 3 min, and vortexed briefly. The samples were then centrifuged at 12,000 rpm for 30 min at 4 °C, and the supernatants were transferred to new Eppendorf tubes. For the experimental sample set, each test compound was added to a final concentration of 100 μM; for the control sample set, the same volume of vehicle solvent was added. The samples were heated at 37 °C for 1 h and dispensed to 100 μL aliquots. Pairs consisting of one control aliquot and one experimental aliquot were heated at 40 °C, 43 °C, 46 °C, 49 °C, 52 °C, 55 °C, 58 °C, 61 °C, or 67 °C for 3 min. Finally, the samples were placed on ice and subjected to western blot analysis using antisera to tNOX [18,61].