Figure 8. Exposure to H₂O₂ causes ubiquitination of Mfn2.

Control fibroblasts were cultured under basal conditions, treated with 1 µM valinomycin for 6 h or with 100 µM H₂O₂ for 12 h. Cells were harvested and mitochondrial and cytosolic fractions were analyzed by Western blotting. (A) The subcellular localization of Mfn2 and Parkin was determined. Quality of cellular fractionation was confirmed using antibodies against VDAC1 and β-actin. (B) Densitometric analysis of the Western blot results revealed a significant drop in protein levels of non-modified Mfn2 (normalized against VDAC1 expression level) in the mitochondrial fraction after valinomycin or H₂O₂ treatment. (C) Furthermore, both treatments caused a significant drop in protein levels of Parkin (normalized against β-actin expression) in the cytosolic fraction. (A, right panel) A longer exposure of the Western blot shows mitochondrial translocation of Parkin after both treatments. For quantification of protein levels, blots of three independent experiments were evaluated. In the graphs mean intensities +/− standard deviation are given. Cyt – cytosolic fraction; H₂O₂ – hydrogen peroxide; Mit – mitochondrial fraction; Mfn2 – mitofusin 2; Ub-Mfn2 – ubiquitinated mitofusin 2; Val – Valinomycin; VDAC1 – voltage-dependent anion channel 1.