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. 2018 Feb 7;315(4):F781–F790. doi: 10.1152/ajprenal.00485.2017

Fig. 5.

Fig. 5.

WNK4 promotes NKCC2 phosphorylation in HEK293 cells. A: HEK293 cells were transiently transfected with NKCC2-Flag and SPAK-HA with either WNK4-HA or WNK4-L319F-HA (constitutively active). The effect of WNK4 coexpression on NKCC2 phosphorylation was assessed by Western blot using anti-NKCC2 (MRC PPU, Dundee Univ. S838B) and anti-pS91-NKCC2 (MRC PPU, Dundee Univ. S451C). Prior to blotting, NKCC2 was immunoprecipitated to ensure the specificity of the signal obtained with the phosphoantibody. As a control, a set of HEK293 cells transfected with NKCC2-Flag and SPAK-HA were treated with basic medium (isotonic, normal-[Cl−] medium) or hypotonic/low-chloride medium (HLC), which induces NKCC2 phosphorylation (1). B: results of quantitation. Densitometric analysis was performed using ImageJ. *P < 0.05 vs. empty; n = 4.