Figure 2: 9030617O03Rik was located in the mitochondrial matrix and was attached to inner membrane.

(a) (Pro K) protection assays show target protein locations in mitochondria. Isolated mouse liver mitochondria were incubated in hypotonic buffer or with Triton-X and were then treated with Proteinase K. Samples were then subjected to immunoblotting using antibodies against 9030617O03Rik or individual proteins. Tom20, Opa1, and GRP75 were used as markers of the outer membrane, inner membrane, and matrix, respectively. (b) Alkali/Salt extraction assays show strengths of target protein attachment to membranes. Isolated mouse liver mitochondria samples were subjected to 0.1 M Na₂CO₃ or 0.5 M NaCl and were divided into pellets (P) and supernatant (S). (c) Representative images of H9C2 rat cardiomyoblast cells transfected with various plasmids; cells were stained with anti- 4′,6-diamidino-2-phenylindole (DAPI) (blue) to visualize nuclei, anti-Tom20 (green) to visualize mitochondria, and anti-hemagglutinin (HA) (red) to visualize the expressed proteins; original magnification, x1,000.