FIGURE 4.

Expression of WNK1 increases the association of endogenous TBC1D4 with 14-3-3 proteins. HEK293 cells were transfected with either GFP empty vector, or fragment GFP-WNK1(1–538) or its respective kinase-dead mutant. Endogenous TBC1D4 was immunoprecipitated from cell lysates prepared under nondenaturing conditions and probed by Western blot (WB). Shown are the levels of GFP-WNK1 and endogenous TBC1D4 and 14-3-3 proteins in total cell lysates (Input; left panel) and in the immunoprecipitate (IP; right panel). The quantification of the 14-3-3 band intensities is given below the blot as fold over control values. Note that the amount of 14-3-3 co-precipitating with TBC1D4 increased considerably in the presence of WNK1, not, however, of kinase-dead WNK1.