105.251 qs cuvettes

All FRET assays were conducted at 4°C using a QuantaMaster QM4CW (PTI) fluorimeter and Hellma 105.251-QS cuvettes. Time courses followed the donor channel using an excitation of 555 nm and emission of 570 nm. Recordings were taken every 30 s for 2 hrs once 100 nM heterolabeled KEAP1 was mixed with 5 uM of chase protein. GroEL was obtained from Sigma-Aldrich (C7688). Overnight incubations were performed by taking spectra (555 nm excitation) of 100 nM heterolabeled KEAP1 dimer, followed by an overnight 4°C incubation with 5 μM of chase protein. Similar measurements were performed for KEAP1 dimers labeled with either FRET donor or acceptor and then mixed. The bar graphs depicting percent increase in donor fluorescence upon addition of FBXL17, FBXL17^ΔCTH, or KEAP1^F64A were calculated from emission at 570nm. Calculations corrected for the dilution upon adding the chase protein.