3.2 Compound 270 inhibits NF-κB transcriptional activity and
directly interacts with NF-κB in HEK 293 and RAW 264.7 cells
CETSA method directly detects the interaction between the drug molecule
and target protein in cells, as the binding of molecule can increase the
thermal stability of protein (Martinez
Molina et al. , 2013).Thus we investigated the binding ability of
270 to NF-κB in HEK 293 cells (transfected with an NF-kB expression
plasmid) and RAW 264.7 macrophages. Cells were incubated separately with
DMSO and 270 for 3 h. Then cells were heated at the indicated
temperatures and harvested cells were analyzed by western blotting. Our
results indicated that 270 treatment protected NF-κB protein from
destabilization in HEK 293 cells (transfected with an NF-kB expression
plasmid) (Figure 2C,D) and RAW 264.7 macrophages (Figure 2E,F) compared
to DMSO, suggesting that 270 directly interacts with NF-κB proteins.