siRNA-mediated down-regulation of WNK1/OSR1 attenuates the TMZ-induced NKCC1 activation. A. Representative immunoblots showing effects of WNK1 siRNA treatment on expression of t-WNK1 (upper panel) and p-NKCC1, p-OSR1 and p-SPAK (lower panel). C. Representative immunoblots showing effects of OSR1 siRNA treatment on expression of t-OSR1 (upper panel) and p-NKCC1 (lower panel). GC#99 were treated with either scramble siRNA (Scr), WNK1 siRNA, or OSR1 siRNA for 48 h, followed by exposure to control medium (Con), 10 μM BMT, 100 μM TMZ, or 100 μM TMZ plus 10 μM BMT (T + B) for 4 h. B and D. Summary data of immunoblotting. Left panel, the expression level of each phosphorylated protein was normalized by its total protein and then the basal expression under control conditions. Right panel, the expression level of each total protein was normalized by α-tubulin and then the basal expression in Scr-treated cells under control conditions. Data are mean ± SEM, n = 3 ~ 5, *p < 0.05 vs. Con, #p < 0.05 vs. Scr.