Fig. 6. Endogenous NCC phosphorylation in the mpkDCT cell line. (A) Total cell extracts derived from mpkDCT, HEK293, mouse embryonic fibroblast (MEF) or embryonic stem (ES) cells were immunoblotted with a total NCC antibody from Chemicon (AB3553). Moesin levels were monitored as a loading control. (B,C) mpkDCT cells were treated with either basic (–) or hypotonic low-chloride (+) medium for 30 minutes and lysed. The activity and phosphorylation state of endogenous WNK1 and SPAK/OSR1 were analysed as described in the legend to Fig. 3A,B. Results of duplicate samples are shown and similar results were obtained in two separate experiments. (D) Phosphorylation of endogenous NCC in cell extracts from basic (–) and hypotonic low-chloride (+)-treated mpkDCT cells was determined by immunoblot analysis with the indicated total and phospho-NCC antibodies. Results of duplicate samples are shown and similar results were obtained in two separate experiments.