CETSA was performed as described (78). Briefly, HEK293T cells were treated with TSI-13-57 or DMSO as control at 37°C with 5% CO2 for 60 min in cell culture dishes. Cells were resuspended in medium and spun down at 240g for 5 min at room temperature. Medium was carefully removed, and cells were resuspended in PBS. Cells were spun down again at 240g for 5 min at room temperature. PBS was carefully removed, and the cell pellet was resuspended in PBS supplemented with protease inhibitors to obtain a cell density of 8 × 106 cells/ml. The cell suspension (0.8 million in 100 μl of volume) was transferred to multiple tubes in a real-time polymerase chain reaction (PCR) plate (Applied Biosystems). The PCR plate was loaded to the heating block of a PTC-200 Gradient Thermocycler (MJ Research) at 25°C. Samples were heated to their desired temperatures in parallel by applying a temperature gradient covering a range between 40° and 64°C. The respective temperatures were maintained for 3 min before the samples were cooled and maintained at 25°C for 3 min. Next, the tubes were immediately shock-frozen in liquid nitrogen. Cells were lysed by two alternating thaw-freeze cycles in a heating block (25°C) and in liquid nitrogen, respectively. The resulting suspensions were centrifuged at 20,000g for 20 min at 4°C. For the following steps the lysates were kept on ice. Supernatants of each sample were carefully transferred to reaction tubes without touching or disturbing the pellets and were analyzed by SDS-PAGE, followed by immunoblot analysis.

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