Fig. 7. Western blot analyses of proteins extracted from a different number of cells and subcellular fractions, using the anti-Dd-EF2H antibody. (A,B) Cell lysates prepared from the indicated number of cells were applied to lanes of 10% SDS-PAGE, followed by western blotting. (C) Ax-2 cells were fractionated to cytosolic (Cyt) and mitochondria-rich (Mit) fractions. Vegetative cells were harvested at the mid-late exponential growth phase and pelleted by centrifugation (400 g, 1 minute). The pellet was suspended in ice-cold 50 mM Tris-HCl buffer (pH 7.5) containing 2.5 M sucrose and homogenized using a glass homogenizer. The homogenate was centrifuged for 10 minutes at 900 g to remove undisrupted cells and nuclei, and the resulting supernatant was centrifuged for 20 minutes at 2000 g. The pellet thus obtained was used as a Mit fraction. The supernatant was again centrifuged for 1 hour at 16,000 g, and the supernatant was put to use as a Cyt fraction. All of the processes were carried out around 4°C. In the Mit fraction, a band of 71 kDa protein can be detected in addition to contaminated 101 kDa Dd-EF2H. Based on the band patterns observed, it is most likely that the 58 kDa and 36 kDa proteins in B are hydrolysis products of 101 kDa Dd-EF2H, while the 41 kDa protein may be a hydrolysis product of the mitochondrial 71 kDa protein.