Fig. 8. Functional assays revealed that ALD increases the K+ efflux of NKCC1. The functionality of NKCC1 activity was analyzed in individual cells using bumetanide, an NKCC1 antagonist, combined with thallium (TI+) uptake, a physiological measure of NKCC1 activity. Cells were loaded with thallium-sensitive dye FluxOR. Fluorescence photos (fluorescence excitation and emission: 488/525 nm) were taken 90 s after addition of 2.8 mM TISO4 to the media. Top row: photomicrographs represent the fluorescence signal average after application of bumetanide, from left to right: 1) control: no bumetanide; 2) ALD: aldosterone alone, no bumetanide; 3) MG132: the proteasome inhibitor alone (no bumetanide); and 4) MG132 in combination with ALD (no bumetanide). Bottom row: photomicrographs represent the fluorescence signal average after application of bumetanide, from left to right: 1) control: with bumetanide; 2) ALD: aldosterone alone, with bumetanide; 3) MG132 alone (with bumetanide); and 4) MG132 combination with ALD (with bumetanide). Bar graph (bottom) represents the bumetanide-sensitive component of the TI+ uptake, as measured with densitometry, in 24 labeled cells for each condition. The intensity reported on the ordinate in the bar graph is normalized by subtracting the bumetanide fluorescence levels (bottom row) from the nonbumetanide conditions (top row). ***P < 0.001. DOI: (10.1152/ajpcell.00096.2013)