Fig. 3.

With no lysine kinase (WNK) 3 activity is modulated by cell volume but not by intracellular chloride concentration ([Cl⁻]_i), whereas WNK4 activity is modulated by [Cl⁻]_i but not by cell volume. Representative Western blot assay (top) and densitometric analysis (bottom) are shown for experiments that used a phosphoantibody against p-Ser³⁸²-WNK1 (p-WNK) (25), which detects WNK3 and WNK4 phosphorylation at Ser³⁰⁸ and Ser³⁷⁹, respectively; phosphorylation of the proteins was used as surrogates of WNK activity. cMyc-WNK3, HA-WNK4, and β-actin levels were used as total protein loading control. Total proteins were extracted from X. laevis oocytes injected with wild-type hWNK3 (WNK3; A), wild-type mWNK4 (WNK4; B), or the chloride-binding motif mutant mWNK4-L319F (C) cRNAs; oocytes were previously incubated in hypotonic media (46 mM sodium chloride, 150 mOsm), isotonic media (46 mM sodium chloride, 200 mOsm), and hypertonic media (46 mM sodium chloride, 250 mOsm) for 1 h (see materials and methods). Low-chloride hypotonic stress (LCHS) incubation was used as control group for wild-type WNK4 (B) and mutant WNK4-L319F phosphorylation (C). As shown in A, WNK3 phosphorylation was modulated by extracellular tonicity, since all groups were exposed to the same concentration of extracellular chloride. B and C show no significant difference between tonicity conditions regarding WNK4 phosphorylation. Densitometric analysis used WNK phosphorylation/total protein under isotonic conditions as 100% (an arbitrary unit), and tonicity conditions were normalized accordingly. Data are the means ± SE of at least 4 different experiments (*P < 0.01 vs. isotonic bar).