Western blot-based CETSA

CCRF-CEM cells were counted, centrifuged at 300 × g for 5 min, and resuspended into new media at a dilution of 0.8 × 10⁶ cells/ml. Cells were aliquoted into 15 ml of cell suspension per standing 25-cm² flask and incubated at 37 °C and 5% CO₂. The next day MQ was added into the media to a final concentration of 10 or 15 µM of MQ and incubated for 2 h at 37 °C and 5% CO₂; control cells were left untreated. After 2 h, cells were collected and centrifuged for 3 min at 300 × g, washed with HBSS, and counted by trypan blue and the cell counter. Cells were centrifuged for 3 min at 300 × g, resuspended to 60 × 10⁶ cells/ml in HBSS, and aliquoted into 50 µl/PCR tubes into 7 different PCR stripes (7 different temperatures). The PCR stripes were transferred to PCR machine Veriti thermal cycler (Applied Biosystems) and heated for 3 min at the indicated temperatures (37–65 °C) after which the samples were cooled down on ice and 0.5 µl of 100× Protease Inhibitor Cocktail in DMSO (P8340, Merck/Sigma-Aldrich) was added to each sample. Samples were vortexed and snap frozen by liquid nitrogen and then thawed in 20 °C in the PCR machine; this was repeated twice. Lysates were centrifuged at 20,000 × g for 20 min at 4 °C and then 40 µl of the supernatant was moved to new PCR tubes and were frozen at −80 °C.