Fig. 5. SPAK/OSR1 interaction with NCC mediates NCC phosphorylation. (A) HEK293 cells were transfected with constructs encoding wild-type (WT) or indicated mutants of human FLAG-NCC. At 36 hours post-transfection, cells were lysed, and NCC was immunoprecipitated and subjected to immunoblot analysis with the indicated antibodies. Similar results were obtained in two separate experiments. (B) As above, except that cells were treated with either basic (–) or hypotonic low-chloride (+) medium for 30 minutes and lysed. Cell lysates were subjected to immunoblot analysis with the indicated antibodies.