Fig. 2. Subcellular localization of rTOM70 in rat liver cells. (A) Comparison of SDS-PAGE profiles of mitochondrial authentic OM70 (Mt) and the in vitro synthesized OM70 (TL) as detected by autoradiography and immunoblotting, respectively. (B) 50 µg each of mitochondria (mit), microsomes (ms) and cytosol (cyt) proteins from the rat liver were subjected to SDS-PAGE and immunoblotting using antibodies against the indicated proteins. The immunoreacted bands were visualized by ECL (Amersham). (C) Immunofluorescence detection of TOM70 in HeLa cells. HeLa cells were incubated with MitoTracker at 37°C for 20 minutes, fixed, stained with rabbit IgGs against rTOM70 and then stained with FITC-conjugated goat antibodies against rabbit. Images were obtained and analyzed using confocal fluorescence microscopy. (D) Submitochondrial localization of rTOM70. Submitochondrial fractionation by sucrose density gradient centrifugation was performed as described in the Materials and Methods. After centrifugation, the samples were fractionated from the top of the tubes. The fractions were TCA-precipitated and subjected to SDS-PAGE and then the proteins were detected by immunoblotting with antibodies against the indicated proteins. Inp represents 20 µg of the unfractionated sample.