Cellular thermal shift assay (CETSA)
CETSA can directly detect the binding affinity of drugs and target
proteins in cells or tissues. Briefly, cells were seeded in 10 cm plates
and grown to 80%-90% confluence. The cells were treated with different
concentrations of AZM or dimethyl sulfoxide for 1 hour. After treatment,
the cells were trypsinized, washed twice with PBS, and then resuspended
in PBS solution containing protease inhibitors, and heated at 42 °C, 46
°C, 49 °C, 52 °C, 54 °C, and 57 °C for 3 minutes. Then, following
freezing and thawing, the samples were centrifuged at 12,000 rpm for 20
min at 4 °C and analyzed by western blot.