FIGURE 7.

ROCK is an essential upstream molecule in LIPUS-induced Cot/Tpl2 and ERK activation. A, 3T3-L1, MC3T3-E1, and ST2 cells were pretreated with 1, 2.5, 5, or 10 μm Y-27632 (a ROCK-specific inhibitor) for 1 h followed by LIPUS stimulation for 20 min. Cell lysates were separated by SDS-PAGE, and levels of phosphorylated and total ERK proteins were determined by Western blotting. B, 3T3-L1, MC3T3-E1, and ST2 cells were pretreated with 5 μm Y-27632 for 1 h. Levels of phosphorylated and total Cot/Tpl2 proteins were determined as in A. C and D, 3T3-L1, MC3T3-E1, and ST2 cells were transiently transfected with either ROCK1 siRNA or control siRNA. The inhibitory effects of ROCK1 siRNA on ROCK1 protein expression were confirmed by Western blotting (C). Phosphorylation of Cot/Tpl2 and ERK was analyzed by Western blotting (D). mW, milliwatts.