Figure 2. Assessment of RNAi screen hits on volume-regulated KCC3 and NKCC1 phosphorylation.

(A) Assessment of candidate kinase WNK3. We validated secondary siRNA screen hits using cell lines harboring specific knockout (KO) of the target gene of interest to avoid off-target effects of siRNA. WT and WNK3 KO cells⁶⁰ were incubated 30 min in the absence or presence of STOCK1S-50699 (IN), a SPAK conserved carboxy-terminal (CCT) docking domain inhibitor (see Methods). The lysates were immunoprecipitated (IP) and/or immunoblotted (IB) with the indicated antibodies (see also Supplementary Figure 1A,B). The ratio (KCC3 P-Thr⁹⁹¹)/(total KCC3) ratio was calculated for each kinase KO lysate. Ratios were compared by unpaired t-test (n = 3, mean ± SD). ***p < 0.001; **p < 0.01; *p < 0.05; ns, non-significant. (B) Assessment of candidate kinase PDK1. WT and PDK1 KO cells⁷⁴ were treated and analyzed as in (A). See also Supplementary Figure 2A,C,D. (C) Assessment of candidate kinases AKT1/SGK1. TSC1 or TSC2 KO cells were used as models of down-regulated AKT1 and SGK1 activity⁷⁵, and treated and analyzed as in (A). See also Supplementary Figure 2B,C.