Cellular thermal shift assay (CETSA)

CETSA was performed as described [66]. Shortly, HEK-293T cells in six wells were transfected with plasmids expressing Nsp14-strep and/or SIRT5. After 48 hours, cells were harvested, washed with PBS, and resuspended in PBS supplemented with EDTA-free complete protease inhibitor cocktail (Roche). Intact cells were divided into 100-μl aliquots and heated individually at different temperatures for 3 minutes in a PCR machine (Biorad), followed by cooling for 2 minutes at room temperature. Cell suspensions were freeze-thawed three times with liquid nitrogen, and the soluble fraction was separated from the cell debris by centrifugation at 20,000 × g for 20 minutes at 4°C. Supernatants containing soluble proteins were transferred to new microcentrifuge tubes and analyzed by western blot.