Quantification of Ubiquitylated WNK4 Protein

Western blotting was performed as described (47 (link)). Plasma membrane fraction was purified using plasma membrane isolation kit (Invent biotechnologies). Enrichment in plasma proteins was confirmed in the laboratory (54 (link)). Equal amounts of protein were mixed with Laemmli sample buffer, separated on polyacrylamide gel, and transferred to nitrocellulose membrane. The membrane was incubated with primary and peroxidase-conjugated secondary antibodies, followed by imaging using ECL reagents (Perkin-Elmer). Tubulin and Ponceau S staining were used to ensure equal loading and transfer of samples. For the detection of ubiquitylated WNK4, WNK4-HA was purified by HA- immunoprecipitation (IP) from the cell lysates, and the ubiquitylated WNK4 was detected by Western blotting using anti-ubiquitin antibody (47 (link)). Monoclonal mouse antibody against KLHL3^S433-P was created in the laboratory as described (35 (link)) and was further characterized in this study. Rabbit polyclonal antibodies against NCC phosphorylated at T53 is kindly provided by Johannes Loffing, University of Zurich, Zurich. Other antibodies include anti-KLHL3 (19 (link)), anti-FLAG, anti-tubulin (all from Sigma), anti-WNK4 (created in the laboratory) (35 (link)), anti-phospho SPAK/OSR1, anti-NCC (all from Millipore), anti-total SPAK, anti-ubiquitin, anti-WNK1 (55 (link)), and anti-calcineurin A (Cell Signaling).