Fig. 10.

Inhibitory effect of L-WNK1-Δ11 correlates with KCC3a phosphorylation status in HEK-293 cells A: HEK-293 cells were transiently transfected with full-length wild-type Flag-KCC3a with or without c-Myc-L-WNK1-Δ11. Thirty-six hours after transfection, cells were preincubated in either isotonic or hypotonic Na⁺ and Cl⁻-free medium during 30 min and later transferred to either isotonic or hypotonic Na⁺-free medium for another 10 min. Cells were lysed, and total cell extracts were immunoblotted using specific antibodies against total KCC3a, pT991 and pT1048 of KCC3a and GAPDH as loading control. B: densitometric analysis of data compiled from 3 different experiments. KCC3a basal isotonic phosphorylation (white bar) was arbitrarily set as 100% and the corresponding groups were normalized accordingly. The result of densitometric analysis is expressed as the percentage of pT991 (iso or hypo) over total KCC3a (iso or hypo) and the percentage of pT1048 (iso or hypo) over total KCC3a (iso or hypo). P = NS by Mann-Whitney test; n = 3.