Skip to main content
. Author manuscript; available in PMC: 2016 Oct 3.
Published in final edited form as: Am J Physiol Cell Physiol. 2008 Dec 24;296(3):C463–C475. doi: 10.1152/ajpcell.00467.2008

Fig. 1.

Fig. 1

Hyperosmolarity induces rapid and reversible cofilin phosphorylation in kidney tubular cells. A: confluent layers of LLC-PK1 proximal tubule cells grown in 6-well plates were preincubated in isotonic (Iso) medium for 10 min and then exposed to iso- or hyperosmolarity (Hyper = isotonic medium supplemented with 300 mM sucrose) for the indicated times. Cells were then lysed, and aliquots of cell lysates containing equal amounts of protein were subjected to Western blot analysis using an anti-phospho-cofilin (pcof) antibody. To check for total cofilin expression and the equality of protein loading, the blots were reprobed using antibodies against cofilin (cof) and actin. Blot is not shown for the isotonic full time course. Routinely a 5-min isotonic treatment was used as control (labeled as Iso), since maximal responses (plateau) to hyperosmolarity were usually attained at this time point. Bottom: densitometric analysis of the hyperosmolarity-induced cof phosphorylation. Data are expressed as fold change, normalized to the pcof-to-cof ratio of the isotonic sample as 1. Means ± SE for n = 12 separate experiments are shown. B: reversibility of the osmotically provoked cof phosphorylation. Cells were treated iso- or hypertonically for 5 min as in A and then isotonicity was reestablished for the indicated times (min). Subsequently cells were lysed and processed as in A. C: dependence of the hypertonicity-induced cof phosphorylation on the osmotic concentration of the medium. Osmolarity was set to the indicated values using various concentrations of sucrose added to the isotonic medium. After 10 min preincubation in the isotonic medium, cells were challenged with the corresponding level of osmolarity for 5 min and then processed as in A. Changes in pcof levels are expressed compared with isotonic control. D: Madin-Darby canine kidney (MDCK) distal tubular cells exhibit similar cof phosphorylation upon hyperosmotic treatment. Similar conditions were used as in A. E: hyperosmolarity does not increase cof phosphorylation in Chinese hamster ovary (CHO) cells.