Figure 5. Effects of phosphatase and kinase inhibitors on phosphorylation of fNKCC2A and fNKCC1.

Experiments were carried out as described in Figure 4, but without the addition of ⁸⁶Rb⁺. Cell lysates were subjected to SDS-PAGE, immunoblotted, and probed with antibodies against phospho-NKCC1 and 2 (NKCC-P, R5), NKCC2 (anti-NKCC2), NKCC1 and 2 (T4) and NKCC1 (N1). Representative blots are shown with control and drug-treated samples from the same blot. Estimated molecular mass is indicated to the right of each panel. (A) Effect of 0.25 µM calyculin A on monomeric NKCC. An approximate 20 kDa band-shift in calyculin-treated cells is only observed with the phospho-specific antibody R5 (arrow) but not with NKCC-P. (B) High molecular mass bands in calyculin A treated fNKCC2A cells. (C) Example of 20 kDa band-shift (arrow) in HEK-293 cells. Cells had been pre-incubated in basic medium, a Na⁺-free medium or with calyculin. (D) Effect of 50 µM PP1 and 120 µM genistein on monomeric NKCC. (E) High molecular mass bands in PP1 or genistein treated fNKCC2A cells.