Cellular Thermal Shift Assay (CETSA)

This assay was performed as previously described (Martinez Molina et al., 2013; Martinez Molina and Nordlund, 2016). In brief, cells were trypsinized and washed with PBS. The cell lysates were diluted with appropriate buffer and divided into aliquots that were treated with CDDO-me (1 µM). After 30 min incubation at room temperature, the respective lysates were divided into smaller (50 µL) aliquots and heated individually at different temperatures for 3 min (Veriti Thermal Cycler, Applied Biosystems, Foster City, CA, USA) followed by cooling for 3 min at room temperature. The heated lysates were centrifuged at 20,000 × g for 20 min at 4°C to separate the soluble fractions from precipitates. The supernatants were transferred to new tubes and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting.