Probing KEAP1-MCB-613 Interactions

293FT cells were transfected with HA Clover KEAP1 WT as described above. After 48 hours, the cells were washed once in ice-cold PBS and collected in ice-cold RIPA buffer (Sigma). The cells were incubated on ice for 30 minutes with intermittent vortexing, and the resultant lysates subjected to centrifugation at 10,000 rpm for 10 minutes at 4 °C. A fraction of the supernatant was reserved for input and prepared for western blotting as described above. The remaining supernatant was exposed to GFP-Trap agarose beads (Proteintech) and rotated end-over-end at 4 °C overnight before washing 5 times with ice-cold RIPA buffer. The beads were then equilibrated in room-temperature binding buffer (25 mM Tris (pH 7.5), 100 mM NaCl, 0.1% NP-40, 1 mM DTT, 5% glycerol). 10 μg of purified His6-KEAP1 was added and the samples were rotated end-over-end at room temperature for 15 minutes. MCB-613 (2, 20, or 200 μM) was added and the samples were rotated for another 15 minutes. Samples were washed 3 times in room-temperature binding buffer, re-suspended in 80 μl of NuPAGE LDS Sample Buffer (4X), boiled at 95 °C for 5 minutes, and analyzed by western blot. Targets were detected using anti-GFP (#2555, CST) and anti-His (#2365, CST) primary antibodies.