Fig. 3. Circadian regulation of the WNK/OXSR1/SLC12A pathway activity.

a Schematic of the WNK/OXSR1 pathway and regulation of the N(K)CC and KCC transporters. b In vitro kinase activity assays for purified WNK1 and 3-Phosphoinositide Dependent Protein Kinase 1 (PDPK1) upon increasing concentrations of NaCl or polyethylene glycol (PEG) (n = 1). WNK1 but not PDPK1 is sensitive to increased macromolecular crowding (mimicked by PEG). Note that WNK1 is inhibited by high concentrations of Cl⁻. c Phosphorylation of cellular OXSR1 upon hypotonic treatment and serum, indicating that decreased intracellular Cl⁻ and increased cytosolic crowding lead to OXSR1 phosphorylation (n = 1). d Representative immunoblots and quantification showing phospho-OXSR1 and OXSR1 abundance in fibroblasts sampled every 3 h for 3 days in constant conditions (n = 3). The asterisk indicates the P-OXSR1 band. p value indicates comparison of damped cosine wave with straight-line fit (null hypothesis = no rhythm). e Model prediction and data showing the difference in cytosolic K⁺ (digitonin-extracted lysates) between cells treated with DIOA (KCC inhibitor) or bumetanide (NKCC inhibitor) compared with vehicle-treated cells at the indicated time points. Cells were treated for 15 min before sampling (n = 4). Statistical test used was two-way ANOVA with Sidak’s MCT. Mean ± SEM shown throughout. Cell type used was lung fibroblasts (c–e).