Fig. 6.

Hyperosmolarity induces ROCK-mediated LIM kinase and cof phosphorylation. A and B: cells grown in 6-well plates were preincubated for 10 min with vehicle or 10 μM Y-27632 in isotonic medium and then challenged with hyperosmolarity (300 mM sucrose) in the presence or absence of Y-27632 for the indicated times. Subsequently cells were lysed and the lysates were subjected to Western blot anaylsis using pcof and cof antibodies. Cumulated data for n = 6. B: similar experiments were performed as in A, and the lysates were probed with phospho-LIMK and LIMK2 antibodies (n = 4). Blotting with anti-LIMK1 did not provide clear, specific labeling (not shown). C: cells were preincubated with vehicle or 10 μM PAK18, a selective PAK inhibitor, for 20 min under isotonoic condition and then challenged with hyperosmolarity for 5 min in the presence or absence of the drug. Cof and pcof content were determined by Western blots. GADPH was used as a loading control. D, top: cells were pretreated in isotonic medium with or without Y-27632, treated iso-or hypertonically for 5 min (as indicated) in the absence or presence of 10 μM Y-27632, and then processed for Western blotting using an anti-phospho-p38 antibody. The blots were reprobed with anti-p38. Bottom: cells were preincubated with 10 μM SB-203580, treated in the absence or presence of the drug, and processed as stated above. Cell lysates were probed for pcof and reprobed for cof. E: similar experiments were performed as in C, and the lysates were probed with anti-p38 and anti-phospho-p38 antibodies.