Figure 4 of
Fernandes, Mol Vis 2004;
Figure 4. Effect of hyperglycemia on ubiquitinylation
Hyperglycemia altered endogenous ubiquitin conjugates and de novo ubiquitin conjugation activity in diabetic animal (A, B) and in cells exposed to high glucose (C, D). Diabetic animals (A, lanes labeled "GK") or cells exposed to high glucose (C, right) show increased levels of endogenous high molecular weight ubiquitin conjugates. Cells exposed to high glucose also show a significant decrease in the total amount of GLUT1 (C). Retinal tissues or cells were lysed, proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed with antibodies directed against ubiquitin conjugates (FK2). The increase in endogenous ubiquitin conjugates is associated with an increased ability of retinal (B) or cell (D) extracts to conjugate radiolabeled 125I-ubiquitin to endogenous substrates. Conjugation activity was determined either in the presence of an ATP generating buffer ("Buffer ATP" in B and D) or as a control in the absence of ATP ("Buffer A" in B and D). Conjugation experiments were performed for 20 min. Proteins were resolved by SDS-PAGE and the dried gels were used for autoradiography.