Figure 2. Interaction of WNK1/WNK4 with SPAK and OSR1.

(A) Multiple sequence alignment of the indicated STE20 human kinases. Identical residues are highlighted in black and similar residues are highlighted in grey. The kinase domain is marked with a continuous line, regions of non-similarity between SPAK and OSR1 are marked with a dotted line, the Mg²⁺-binding aspartic acid residue mutated to inactivate SPAK and OSR1 is marked with a triangle, the T-loop threonine residue phosphorylated by WNK1/WNK4 is marked with an asterisk, and the non-catalytic serine residue phosphorylated by WNK1/WNK4 with a square. (B) HEK-293 cells were transfected with constructs encoding the indicated GST-fusion proteins. At 36 h post-transfection, the GST-fusion proteins were affinity-purified and immunoblotted with GST antibodies or WNK1 antibody to detect endogenously associated WNK1. (C) HEK-293 cells were co-transfected with the indicated combinations of FLAG-tagged WNK1/WNK4, GST–SPAK, GST–OSR1 or empty pEBG2T vector. GST-fusion proteins were affinity-purified and immunoblotted with FLAG antibody to detect WNK1 or WNK4 expression or GST antibody to detect SPAK, OSR1 or GST expression.