Fig. 3. Characterization of rTOM70 as a mitochondrial integral membrane protein. (A) Mitochondria were treated with either 100 mM Na2CO3 (pH 11.5) or 1 M KCl at 0°C for 30 minutes. The reaction mixtures were centrifuged to separate the supernatant (S) and precipitate (P) fractions, and each fraction was subjected to SDS-PAGE followed by immunoblot analysis using antibodies against the indicated proteins. (B) Rat liver mitochondria were treated with the indicated concentrations of trypsin at 0°C for 20 minutes and then soybean trypsin inhibitor was added to the reaction mixtures and incubated at 0°C for 20 minutes. The reaction mixtures were centrifuged to separate the supernatant and precipitate fractions, and each fraction was resolved by SDS-PAGE and analyzed by immunoblotting with the antibodies against the indicated proteins.