Differential Scanning Fluorimetry (DSF)

CDK12KD/CCNKΔC (WT or G731E/R) and DDB1ΔBPB were diluted to 5 μM with DSF assay buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP). 50 µM HQ461 or 10 µM HBx was added to diluted proteins (final DMSO concentration 0.25%). Twenty µL of protein-compound mix was aliquoted per qPCR tube (Labtidebiotech, Shanghai, China, P01-0803E) followed by the addition of 5 µL of 1:400 diluted SYPRO Orange (Sigma-Aldrich, MO, USA, S5692). After incubation on ice for 20 min, fluorescence measurements were performed using a Bio-Rad CFX96 Realtime PCR instrument. During the DSF experiment, the temperature was increased from 10°C to 95°C at an increment of 0.5 °C with equilibration time of 10 s at each temperature prior to measurement. Tm was defined as the temperature corresponding to the maximum value of the first derivative of fluorescence transition.