Differences in OPA1 levels are due to protein accessibility. (A) Semiquantitative RT-PCR was performed on mRNA isolated from Omi/HtrA2 WT or KO and re-transfected MEF cells. There are no differences in the OPA1 mRNA levels (upper panel). Amplification of β-actin served as loading control (lower panel). (B) For analysis of the full cell content, 150,000 control and Omi/HtrA2 KO MEF cells were harvested, lysed directly in Laemmli SDS-PAGE sample buffer and analyzed by Western blot. Immunoblots were probed with anti-OPA1 (short and long exposure) and anti-Omi/HtrA2 and β-actin was used as a loading control. (C) Mitochondrial and cytosolic fractions prepared from Omi/HtrA2 WT and KO cells were subjected to Western blotting and analyzed for localization of both OPA1 and Omi/HtrA2. The purity of the cytosolic (Cyto) and mitochondrial (Mito) fractions was assessed by probing for Hsp90 and VDAC1, respectively. (D) Mitochondria isolated from control (left lanes) or Omi/HtrA2 KO (right lanes) MEF cells were subjected to <t>proteinase</t> K digestion for indicated time points. Western blots were prepared and probed with anti-OPA1 and anti-Omi/HtrA2 as indicated. The outer membrane associated protein Fis1 was rapidly degraded whereas the inner membrane protein prohibitin was shielded. The higher molecular weight OPA1 band densitometrically analyzed (arrow head) is indicated and the fold change compared to untreated (time point 0) is shown below the Western blot (D).