Fig. 6. Effects of pH and the MAPK pathway on the destruction of cyclin B in a cell-free preparation. (A) The cell-free preparation at pH 7.0 or 7.3 with or without U0126 (0.1 mM) was incubated for 0, 20, 40, 60 and 85 minutes. After incubation, sample buffer for SDS-PAGE was added to stop the reaction. Then, the samples were analyzed by 10% SDS-PAGE, followed by immunoblotting with an anti-cyclin B antibody. (B) The MAPK activity of the cell-free preparation at pH 7.0 or 7.3 with or without U0126 (0.1 mM). Activation of MAPK was inhibited by U0126. MAPK was activated at pH 7.0 and 7.3 (arrow), and inactivated at pH 7.0 with U0126 (arrowhead). Each sample was analyzed by 12.5% SDS-PAGE, followed by immunoblotting with an anti-MAPK (ERK1) antibody.