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. 2013 Mar 29;288(20):14569–14583. doi: 10.1074/jbc.M112.437392

FIGURE 5.

FIGURE 5.

FRET analysis of SUMO-modified Nrf2 in PML-NBs. HepG2 cells were transfected with pEGFP-Nrf2 (1 μg) along with expression plasmid for RFP-SUMO (0.5 μg). The cells were harvested and fixed as described under “Experimental Procedures.” Individual spots containing PML, GFP-Nrf2, and RFP-SUMO were subjected to FRET analysis. Donor (GFP) and acceptor (RFP) images were acquired with a Nikon A1R laser-scanning confocal microscope, as described under “Experimental Procedures.” A and B, the arrows indicate locations of pre- and post-bleaching. The differential interference contrast (DIC) images are shown in panels 7 and 8. A, FRET analysis of interaction of GFP-Nrf2 with RFP-SUMO-1. B, FRET analysis of interaction of GFP-Nrf2 with RFP-SUMO-2. C, lack of FRET signals between GFP-Nrf2 and nonconjugatable SUMO-1 (RFP-SUMO-1ΔGG). D, quantification of the FRET data. For each treatment, 10–20 cells were imaged. FRET efficiency (%) was calculated from the following equation: FRET efficiency (%) = ((GFPafter − GFPbefore)/GFPafter) × 100 (20). Values plotted are means ± S.E. for n = 17–52. Scale bars, 10 μm.