4.9. Cellular Thermal Shift Assay (CETSA)

HEK293T cells (2 × 10⁶ cells/mL) transfected with HA-TAK1 plasmid for 24 h were divided into two groups and treated with Oe-ME (100 μg/mL) or vehicle (0.2% of DMSO) for 1 h, respectively. The cells were then washed with cold PBS, pelleted, and resuspended in PBS with protease inhibitors. The resuspended cells were thoroughly mixed and aliquoted into PCR tubes (100 μL each) that were then heated at the designated temperature for 3 min (44 °C to 56 °C), cooled at room temperature for 3 min, and finally placed at −70 °C to quickly freeze. The cells were freeze-thawed three times in liquid nitrogen. The resulting cell lysates were centrifuged to isolate the soluble protein from cell debris. The resulting clear supernatant was mixed with a corresponding volume of loading buffer, and the mixture was subjected to immunoblotting as in a previous report [52].