Fig. 9

Qualitative and quantitative analysis of Opa1 and Oma1 protein levels in cells exposed to hypothermia. Protein lysates of porcine aortic endothelial cells (Control, Ctrl) incubated for 3 h, 6 h, 24 h or 48 h at 4 °C, part of them rewarmed for 1 h at 37 °C (e.g. 48/1) were analysed by western blot with antibodies against Opa1 and Oma1. The two heavier bands represent the long isoforms of Opa1 (l-Opa1) known to be involved in inner membrane fusion and the three lighter bands represent the short isoforms (s-Opa1) incapable of mitochondrial fusion (A, E). The transcription inhibitor amanitine (Aman.;10 µg/ml) was added to assess a potential shift to s-Opa1 by missing transcription and thus missing synthesis of l-Opa1 (E, F). Cleavage of Oma1 was analysed (D), high exposure to carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 20 µM) served as positive control. Representative figures of n = 4 experiments. Coomassie staining was used as loading control (coom.). The band intensity was quantified using the BioVision software and plotted as s-Opa1 to total Opa1 (B, F) or total Opa1 content (C). Means ± SD of n = 4 experiments. Cold conditions are shown as grey bars, warm conditions are shown as open, dotted or hatched bars. ** Significantly different to control (p ≤ 0.01) *** (p ≤ 0.005)