KO Cell Lines

WNK1 gene knockout cell lines provide an effective method for in-depth study of the WNK signaling pathway and its role in physiological processes such as ion transport and volume regulation(A Roy, et al.,2015)

(A) Whole cell lysates (20ug) from WT and WNKl KO clone 4 cells were probed for WNK-SPAK/OSRl pathway components by immunoblotting following a 30 min exposure to isotonic control media or media containing 0.5M sorbitol. (B) Quantification of immunoblots for WNK kinases in (A). In WNKl KO cells, the abundance of WNK2, WNK3, and OSRl increased significantly following exposure to hypertonicity, approximating their protein levels in WT cells subjected to the same condition. In contrast, the abundance of WNK4 and phosphorylated SPAK/OSRl were lower in sorbitol. exposed WNKl KO cells compared to wild type.(n=4 for all samples; *p<0.05, **p<0.01, or***p<0.001 by one-way ANOVA, Tukey's multiple comparison post-hoc test).(C)Average changes in relative cell volume were determined in WT and WNKl KO cells (clone 4) with the calcein method (20). RV after a 20-min of exposure to 0.5M sorbitol in HEPES buffer (800 mOsm) was detected in WNKl WT cells. Each tracing represents measurements taken in 60 cells across 3 separate experiments. (D) Effects of bumetanide (BMT, 10 μM) on RVl. The “WT +BMT” tracing (open circles)represents measurements taken from 20 cells across a single experiment. (E). Lack of RVl in WNKl KO cells. The “WNKI KO + BMT” tracing (open circles) represents measurements taken from 20 cells across a single experiment.