Fig. 4. NCC phosphorylation-site characterisation employing phosphospecific antibodies. (A) HEK293 cells were transfected with wild-type (WT) human FLAG-NCC or the indicated mutant forms of NCC. At 36 hours post-transfection, cells were treated with either basic (–) or hypotonic low-chloride (+) medium for 30 minutes and lysed. Total cell extracts were immunoblotted with NCC-phosphospecific and total antibodies. Similar results were obtained in two separate experiments. AAAA corresponds to a quadruple NCC[1-100] mutant in which Thr46, Thr55, Thr60 and Ser91 are changed to Ala. (B) Wild-type or indicated mutants of NCC[1-100] were phosphorylated with active (Act) or kinase-inactive (KI) mutants of OSR1 or SPAK and phosphorylation analysed by immunoblot analysis. Results of duplicate samples are shown. AAA corresponds to a triple NCC[1-100] mutant in which Thr46, Thr55 and Thr60 are changed to Ala; the kinase-inactive and activated mutants of SPAK/OSR1 employed are defined in the legend to Fig. 2A. (C) HEK293 cells were transfected with wild-type NCC. At 36 hours post-transfection, cells were treated with either basic or hypotonic low-chloride medium for the specified durations and lysed. The phosphorylation of WNK1 at Ser382 and total levels of WNK1 protein were analysed after its immunoprecipitation. Phosphorylation and total levels of SPAK/OSR1 and NCC were analysed in total cell extracts.