FIGURE 1:

Pressure effects on cellular assays of endogenous WNKs and full length WNK3. (A) Left, MDAMB231 cells transiently transfected with PCMV-FLAG-KCC2, lysed and immunoblotted with anti-pKCC2 antibodies and quantification of normalized pKCC2 infrared signal. Mean ± SEM, n =  3 (biological replicates). Significance measured using one sample t and Wilcoxon test in Graphpad Prism 9 (right). (B) Malpighian tubules in which Drosophila WNK was knocked down and fl-human WNK3 was expressed with SPAK^D219A (w; UAS-DmWNK^RNAi UAS-WNK3/UAS-SPAK^D219A; c42-GAL4/+) were subjected to pressure by centrifugation, or to a similar 0.75°C temperature change (control). WNK3 activity was estimated from the ratio of pSPAK to tSPAK. The ratio from the centrifuged sample was normalized to control. Mean ± SEM with individual data points for n = 10 independent replicates shown. ****, p < 0.0001, one-sample t test to theoretical mean of one. A sample Western blot is shown.