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. 2016 Jan 6;6:18710. doi: 10.1038/srep18710

Figure 5. WNK4 in mpkDCT cell is phosphorylated at Ser572 in a hypotonic low-chloride stimulation-dependent manner via the p38MAPK-MK2 pathway.

Figure 5

(a) WNK4 Ser572 (Ser575 in human WNK4) phosphorylation is induced by hypotonic low-chloride stimulation. mpkDCT cells were stimulated with hypotonic low-chloride buffer or isotonic buffer for the indicated times. Full-length blots are presented in Supplementary Fig. S10a. (b) Hypotonic low-chloride stimulation-induced WNK4 Ser572 phosphorylation is attenuated by p38MAPK inhibition. mpkDCT cells were pre-treated with DMSO or SB202190 at the indicated concentration for 30 min before stimulation. Cells were then stimulated with hypotonic low-chloride buffer for the indicated times. Full-length blots are presented in Supplementary Fig. S10b. (c) Hypotonic low-chloride stimulation-induced WNK4 Ser572 phosphorylation is attenuated by MK2 inhibition. mpkDCT cells were pre-treated with DMSO or MK-2 inhibitor III at the indicated concentration for 30 min before stimulation. Cells were then stimulated with hypotonic low-chloride buffer for the indicated times. Full-length blots are presented in Supplementary Fig. S10c. (d) Hypotonic low-chloride stimulation-induced WNK4 Ser572 phosphorylation is attenuated by MK2 knockdown. mpkDCT cells were transiently transfected with the indicated siRNA. At 96 h after transfection, cells were stimulated with hypotonic low-chloride buffer for the indicated times. Full-length blots are presented in Supplementary Fig. S11a. (e) Knockdown efficiency of MK2. mpkDCT cells were transiently transfected with the indicated siRNA. At 96 h after transfection, MK2 mRNA levels were analyzed by qRT-PCR. (f) Hypotonic low-chloride stimulation-induced p38MAPK activation and WNK4 Ser572 phosphorylation are not attenuated by ASK3 knockdown. mpkDCT cells were transiently transfected with the indicated siRNA. At 96 h after transfection, cells were stimulated with hypotonic low-chloride buffer for the indicated times. Full-length blots are presented in Supplementary Fig. S11b. (g) Knockdown efficiency of ASK3. mpkDCT cells were transiently transfected with the indicated siRNA. At 96 h after transfection, ASK3 mRNA levels were analyzed by qRT-PCR.