Fig. 5.

Proximity ligation assay (PLA) for β-TrCP and NRF2. HEK293T cells were transfected with expression vectors for NRF2ΔETGE-V5, or β-TrCP insensitive NRF2ΔETGE-6S6A-V5 mutant. After 24 h, cells were incubated in the presence vehicle (VEH) or PHAR (10 μM) for 6 h. Cells were subjected to the PLA assay using rabbit anti-FLAG and mouse anti-V5. Upon PLA reaction, red puncta represent colocalization of β-TrCP and NRF2. A, representative fluorescence images of DAPI, NRF2, FLAG, PLA results and MERGE. B, Quantification of puncta per nucleus of a total of 50 nuclei per condition. Calibration bar = 20 μm ***p < 0.001 vs. vehicle according to a Student's t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)