Figure 7. Cells exposed to oxidative stress show increased ability to conjugate ubiquitin to exogenous substrates

Levels of ubiquitinylated α-lactalbumin following exposure to 40 mU/ml glucose oxidase (GlucOx, 100 μM of H₂O₂) were determined using exogenous radiolabeled ¹²⁵I-α-lactalbumin, in the presence of both proteasome and isopeptidases inhibitors (MG-132 and Ubal) followed by protein separation by SDS-PAGE. The assay was performed either in the absence or presence of an ATP-generating buffer and in the presence of 2 μg/μl of ubiquitin. The ¹²⁵I-α-lactalbumin-ubiquitin conjugates in the cytosolic fraction were visualized by autoradiography. Band intensity was normalized relative to the control (100%). The values presented in the graph correspond to the mean±SE of four independent assays. The asterisk indicates a difference at p<0.05.