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.