Fig. 7.

Inhibition of ubiquitination prevents a TPA-mediated decrease in NCC activity. mDCT cells were grown to confluence, treated with UBEI-41 (U) or vehicle for 15 min, and then treated with 100 nM TPA (T) or vehicle (DMSO, C) for 15 min. A: cells treated as described were labeled with biotin at 4°C. Biotinylated (cell surface) proteins were recovered using streptavidin-agarose and immunoblotted for NCC. NC, no biotin control. Accompanying densitometry is shown (n = 4). *P < 0.05. B: cells treated as above were subsequently incubated in uptake medium with ²²Na⁺ and either vehicle or 1 mM metolazone. Radioactive uptake was determined as in materials and methods. Thiazide-sensitive uptake was calculated as the difference in radiotracer uptake between the metolazone-containing and the metolazone-free groups. TU, TPA+UBEI-41 (n = 6). *P < 0.01.