Fig. 3. Characterisation of WNK1-SPAK/OSR1 activity and identification of in vivo phosphorylation sites on NCC. (A) HEK293 cells were treated with either basic (–) or hypotonic low-chloride (+) medium for 30 minutes. Endogenous WNK1 was immunoprecipitated and assayed employing kinase-inactive OSR1 as a substrate. Phosphorylation of OSR1 was determined after electrophoresis followed by autoradiography of the Colloidal-blue-stained bands corresponding to OSR1. The incorporation of ³²P radioactivity was also quantified and results are presented as the mean activity ± s.d. for duplicate samples. WNK1 immunoprecipitates were also immunoblotted with the indicated antibodies. (B) As above, except that endogenous SPAK and OSR1 were immunoprecipitated employing an antibody that recognises both proteins. Immunoprecipitated SPAK/OSR1 was assayed employing the CATCHtide peptide substrate (Vitari et al., 2006). Results are presented as the mean activity ± s.d. for duplicate samples. Total cell extracts were also immunoblotted with the indicated antibodies. Results of duplicate samples are shown and similar results were obtained in two separate experiments for A and B. (C) HEK293 cells were transfected with a construct expressing human Flag-NCC. At 36 hours post-transfection, cells were stimulated as in A, and FLAG-NCC was immunoprecipitated and electrophoresed on a polyacrylamide gel. The Colloidal-blue-stained bands corresponding to FLAG-NCC were excised and digested with trypsin. Phosphopeptides were identified by combined liquid chromatography-mass spectrometry and tandem mass spectrometry analysis. The figure shows the signal intensity (cps, counts of ions per second detected) versus the m/z (amu, atomic mass units) for the phosphopeptides derived from NCC isolated from control (red) or hypotonic low-chloride (blue) cells.