Fig. 7.
Increased expression of KLHL3 can overcome effects of Cul3Δ403–459 on WNK4 in the presence of WT Cul3. HEK293TCul3-KO cells (A) or HEK293 cells (B) were transfected with myc-WNK4 and either FLAG-tagged WT Cul3 or Cul3Δ403–459 along with increasing amounts of myc-tagged KLHL3 and analyzed by immunoblot. The increased KLHL3 expression only slightly decreased WNK4 protein abundance in HEK293TCul3-KO cells, however, HEK293 cells had a larger decrease in WNK4 which was not significantly different from WT Cul3. Bar graphs depict quantification of KLHL3 and WNK4 protein abundance. Stain-free imaging was used as a loading control. Data represent relative individual values as well as means ± SE. Statistical differences were examined by one-way ANOVA with Tukey’s post hoc analysis. Cul3, cullin 3; HEK, human embryonic kidney; KLHL3, kelch-like 3; WNK, with-no-lysine kinase; WT, wild type.