Figure 3.

CUL3^Δ403–459 is unable to ubiquitylate WNK1 or WNK4 kinases in an in vitro system, and this deficiency cannot be rescued by the presence of CUL3^WT

• A–C In vitro ubiquitylation assays were performed as described in Figure 2, but with the addition of immunoprecipitated WNK1 in (A), or immunoprecipitated over-expressed FLAG-WNK4 in (B, C) (see Materials and Methods). The WNK kinases are modified by CUL3^WT-KLHL3, with the higher molecular weight smear observed in anti-WNK1 and anti-FLAG panels representative of multiple ubiquitin molecules being covalently attached to the WNK protein. CUL3^Δ403–459 is unable to modify WNKs. Samples from the same assay reactions were divided to allow immunodetection of the different protein components modified within the same assay reaction. (C) CUL3^WT, CUL3^Δ403–459 and an equimolar solution CUL3^WT:CUL3^Δ403–459 (1:1 Mix) were incubated with KLHL3 and immunoprecipitated FLAG-WNK4 in ubiquitylation reactions to determine the influence of CUL3^Δ403–459 on the ubiquitylation activity of CUL3^WT. Notably, the presence of CUL3^Δ403–459 does not inhibit WNK ubiquitylation by CUL3^WT.