Figure 4.

Phosphorylation dynamics of NKCC2 in the absence or in the presence of MAL/VIP17. (A) LLC-PK1cells expressing c-NKCC2 alone or in combination with MAL/VIP17 were grown to confluence on 12-well plates, incubated in low Cl⁻ medium or in the presence of 0.5 μM CalA. Cells were then lysed and subjected to Western blotting using R5 antibody. The same nitrocellulose membranes were stripped and reprobed by Western blotting with T4 antibody. Each experimental condition was examined in quadruplicate. (B) The quantitation of phosphorylated c-NKCC2 was determined from three independent experiments by densitometry of phospho-c-NKCC2 bands (R5 signal) normalized for the corresponding c-NKCC2 bands (T4 signal). Student's t test, *p < 0.001, **p < 0.0001. (C) Both control (MOCK) and MAL/VIP17-depleted MDCK cells (MAL/VIP17 KD) were incubated in regular or low Cl⁻ medium. Cells were then lysed and subjected to Western blotting using R5 antibody (p-c-NKCC2). The same nitrocellulose membranes were stripped and reprobed by Western blotting with T4 antibody (c-NKCC2). Each experimental condition was examined in triplicate. (D) The quantitation of phosphorylated c-NKCC2 was determined from three independent experiments by densitometry of phospho-c-NKCC2 bands (p-c-NKCC2) normalized for the corresponding c-NKCC2 bands (c-NKCC2). Student's t test, **p < 0.0001.