Fig. 7. Mutation of Thr60 of NCC prevents thiazide-sensitive hypotonic low-chloride-induced ²²Na uptake in HEK293 cells. HEK293 cells were transfected with pCMV5 empty vector or constructs encoding the indicated wild-type (WT) or mutant forms of human NCC. At 36 hours post-transfection, ²²Na uptake was assessed in control basic or hypotonic low-chloride-treated cells in the absence or presence of 0.1 mM metolazone in the uptake medium, as described in the Materials and Methods. For each transfection construct used, metolazone-sensitive ²²Na uptake (i.e. metolazone-insensitive counts subtracted) is plotted for both basic and hypotonic low-chloride conditions. The results are presented as the mean ²²Na uptake ± s.d. for triplicate samples. Similar results were obtained in three separate experiments. Expression and phosphorylation of NCC proteins were monitored in parallel experiments following immunoblot analysis using the indicated antibodies.