To evaluate the cellular activity of compound 1, we next prepared HeLa cells transiently expressing mSA (cox8 (mitochondrial targeting signal sequence: MTS) -His6-mSA-FLAG, hereafter cox8-mSA-FLAG or mSA) localized to the mitochondrial matrix (Figure ). Selective expression of cox8-mSA-FLAG in mitochondria was confirmed by isolation of the mitochondrial fraction followed by western blotting, which revealed three anti-FLAG-positive bands at 15-20 kDa. It is known that MTS is generally cleaved at the mitochondrial outer membrane or inner membrane when the protein translocates from the cytosol to the mitochondria. We speculated that the largest protein (about 20 kDa) was intact cox8-mSA-FLAG remaining on the outer membrane or in the intermembrane space of the mitochondria. The lowest-molecular-weight band remained mainly in the cytoplasmic fraction, and may consist of defective or misfolded molecules. We considered that the middle band was the desired mitochondrial mSA, and focused on this band in the following experiments (Figure ).