Fig. 2.

The effect of hyperosmolarity on Rac and PAK in LLC-PK1 cells. A: confluent layers of LLC-PK1 cells in 10-cm dishes were challenged with a hypertonic solution for the indicated time. Cells were then lysed and processed for the Rac activity assay as described under MATERIALS AND METHODS. Before the pull-down assay, an aliquot was taken from each lysate to measure total Rac. Note that hyperosmolarity induced a rapid, small, but significant increase in the amount of captured (GTP-bound) Rac, followed by a substantial decrease (n = 4, *P < 0.05 and *** P < 0.001). B: effect of hyperosmolarity on the intracellular localization of Rac. LLC-PK1 cells, grown to confluence on glass coverslips were treated iso- or hypertonically as indicated. Cells were then fixed and stained for Rac using a primary and a Cy-3-labeled secondary antibody. Note that under isotonic condition Rac distribution is primarily cytosolic, exhibiting some vesicular pattern; after exposure to hypertonicity, the most prominent Rac labeling occurs at the cell periphery, whereas the overall cytosolic staining is reduced. We often observed enhanced and polarized Rac labeling in the perinuclear area. C: hyperosmolarity induces transient PAK phosphorylation. Cells were treated as indicated, and cellular lysates were processed for Western blot analysis using a phospho-specific (Ser 142/144) PAK1/2 antibody. Results of densitometric analyses obtained from 3 independent experiments are shown.