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. 2003 Jan 6;100(2):680–684. doi: 10.1073/pnas.242735399

Figure 4.

Figure 4

Coimmunoprecipitation of WNK4 and C terminus of NCCT. (A) Full-length wild-type WNK4 tagged with the myc epitope and the C terminus of NCCT tagged with the V5 epitope were expressed in HEK 293T cells as described in Methods. Cellular extracts were prepared, and immunoprecipitation was performed with anti-V5 antibodies as described in Methods. The precipitated protein was subjected to SDS/PAGE on 4–15% gradient gels, transferred to membrane, and stained with anti-myc antibody. A 170-kDa protein corresponding to myc-tagged WNK4 is detected in immunoprecipitates from cells expressing both NCCT and WNK4, but not in cells transfected with only one or neither tagged protein. (B) The experimental protocol is as in A, except that the Q562E mutation found in PHAII kindred 13 has been introduced into WNK4; this mutant WNK4 is tagged with the HA epitope rather than myc, and staining is with anti-HA antibody. Mutant WNK4 is coprecipitated by anti-V5 antibody only when both tagged NCCT and WNK4 are transfected.