2.8. Cellular thermal shift assay (CETSA)

In the context of molecular biotechnology, Cellular Thermal Shift Assay (CETSA) is utilized to investigate interactions between intracellular proteins and ligands. This method involves subjecting cell lysates to varying temperatures and subsequently conducting Western blotting to assess alterations in protein thermal stability [16]. For the experiment with A549 cells, the procedure involved treating the cells with griffithazanone A (2 μM) for 4 h. Following this treatment, the cells were harvested by centrifugation, resuspended, and the cell lysate was divided into six equal portions. Each lysate was then exposed to different temperatures (45, 50, 55, 60, 65, and 70 °C) for 5 min. Following the heating process, the lysate were rapidly frozen in liquid nitrogen. This freezing and thawing process was repeated three times and then centrifuged for 20 min at 4 °C and 20,000×g. The thermal stability of proteins in the supernatant was evaluated using Western blot analysis.