Fig. 5

ORF6 hinders NRF2 nuclear translocation but not its activation by phosphorylation. NRF2 activation is assessed by immunoblotting analysis on whole cell lysate of (A) HEK-293T cells expressing the wt-ORF6, M₅₈R or ∆61 protein mutants for total NRF2 or serine 40 phosphorylated NRF2 (pNRF2) expression (mid panel). Densitometric analysis of reactive bands is performed by ImageJ software and, after actin normalization, the fold change in protein accumulation is calculated. The activation rate of NRF2 is inferred by the pNRF2/total NRF2 ratio (lower panel). Nuclear translocation of NRF2 (B), either total or pNRF2 (mid panel), is investigated in HEK-293T cells expressing the wt-ORF6, M₅₈R or ∆61 proteins by immunoblotting. Lamin A/C is used as a loading control. Densitometric analysis is performed on data obtained from three independent experiments, and each condition is run in duplicate (n = 6). Graph values are expressed as the mean fold change in band intensities ± standard deviations (SD). The ratio of nuclear to cytoplasmic pNRF2 is shown in (C), evidencing the reduced nuclear translocation of the active transcription factor. Significance is determined with respect to the negative control (Ctr-), transfected with empty plasmid, sample as **p < 0.005; *p < 0.05