The binding potential of compounds against BRD4-1 were assessed by DSF
using a StepOnePlus Real-Time PCR system (Applied Biosystems, Grand Island, NY).
Purified BRD4-1 (4 μM final concentration; 10 mM HEPES (pH7.5), 100 mM
NaCl, and 1 mM DTT) was assayed, in quadruplicates, in a 96-well plate.
Inhibitors were added to a final concentration of 100 μM and 2%
DMSO. Protein Thermal Shift Dye (1:8000; Applied Biosystems, Grand Island, NY)
was used as the fluorescent probe and fluorescence was measured using the ROX
Reporter channel (620 nm). Protein stability was investigated by programing the
thermocycler to increase the temperature from 25 °C to 99 °C
using 0.2 °C increments and 10 s incubations per increment. The
inflection point of the transition curve/melting temperature (Tm) was
calculated using the Boltzmann equation within the Protein Thermal Shift
Software (v.1.1) (Applied Biosystems, Grand Island, NY). (+)-JQ1 (18 (link)) and dinaciclib (8 (link)) were used as controls for strong and weak binders
of BRD4-1, respectively. The ΔTm was calculated by using DMSO
control wells as reference.
using a StepOnePlus Real-Time PCR system (Applied Biosystems, Grand Island, NY).
Purified BRD4-1 (4 μM final concentration; 10 mM HEPES (pH7.5), 100 mM
NaCl, and 1 mM DTT) was assayed, in quadruplicates, in a 96-well plate.
Inhibitors were added to a final concentration of 100 μM and 2%
DMSO. Protein Thermal Shift Dye (1:8000; Applied Biosystems, Grand Island, NY)
was used as the fluorescent probe and fluorescence was measured using the ROX
Reporter channel (620 nm). Protein stability was investigated by programing the
thermocycler to increase the temperature from 25 °C to 99 °C
using 0.2 °C increments and 10 s incubations per increment. The
inflection point of the transition curve/melting temperature (Tm) was
calculated using the Boltzmann equation within the Protein Thermal Shift
Software (v.1.1) (Applied Biosystems, Grand Island, NY). (+)-JQ1 (18 (link)) and dinaciclib (8 (link)) were used as controls for strong and weak binders
of BRD4-1, respectively. The ΔTm was calculated by using DMSO
control wells as reference.
