Cellular Thermal Shift Assay (CETSA)

CETSA (Martinez Molina and Nordlund, 2016) was performed to confirm the cellular interaction between BRD4 and SN-38 in vitro. The K562 cells were collected and suspended in the PBS. Then, cell suspension was freeze-thawed repeatedly for three times using liquid nitrogen, and the supernatant was collected by centrifuge at 20,000 g for 20 min at 4°C. After that, the protein was incubated with SN-38 for 30 min, and divided into six tubes in average. The tube was then heated at indicated temperature for 3 min, and cooled for 3 min. The supernatant was then transferred to a new microtubule after centrifuge at 20,000 g for 20 min at 4°C, and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by western blot analysis.