2.13. Cellular thermal shift assay (CETSA)

SH‐SY5Y cells were incubated with or without andrographolide (1 μM) for 9 hr, and then the cells were collected and subjected to CETSA assay (Jafari et al., 2014). Briefly, incubated cells were equally divided into 10 parts, each part was heated for 3 min under different temperature (43, 46, 49, 52, 55, 58, 61, 64, 67, and 70°C), and then the heated cells were kept at −80°C for 12 hr, then at room temperature for 5 min, and the process repeated one more time. After that, cell lysates were extracted by centrifugation at 20,000× g, 20 min. Levels of DRP1 were assessed by western blot.