Briefly, fresh kidney cortical samples were homogenized Leukemia as above in ice-cold isolation buffer containing fresh protease inhibitors, and crude membranes were obtained by centrifugation at 48,000 g for 1 h at 2��C (Beckman J2�C21M; JA-20 rotor; Beckman Coulter, Fullerton, CA). Pellets were resuspended, homogenized in a Dounce glass homogenizer, and subjected to Mg2+ aggregation by addition of MgCl2 to a final concentration of 15 mM at 4��C for 20 min. Aggregated membranes were removed by centrifugation at 3,000 g for 10 min at 2��C (Beckman Allegra 21R; Beckman Coulter), and the supernatant was subjected to two additional rounds of Mg2+ aggregation as above (pellets discarded), followed by centrifugation at 48,000 g for 30 min at 2��C. The resulting pellet enriched in BBMV was used for Na+/H+ exchange activity experiments and for immunoblotting.

Na+/H+ exchange activity in BBMV. Na+/H+ exchange activity was measured in fresh BBMV with the acridine orange fluorescence quenching method (53) as previously described (6). Briefly, the BBMV pellet obtained as above was rehomogenized in intravesicular solution [280 mM mannitol, 5 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.5] with a Potter-Elvehjem Teflon-glass homogenizer, equilibrated for 2 h at 4��C, centrifuged at 48,000 g, and resuspended with a 27-gauge syringe needle and intravesicular solution to a final protein concentration of 10 mg/ml. Extravesicular solution [120 mM N-methyl-d-glucamine (NMDG)-gluconate, 20 mM HEPES, pH 7.5] with fresh acridine orange (Invitrogen) added to a final concentration of 6 ��M was loaded in a cuvette equipped with a magnetic mini stir bar.

Acridine orange fluorescence was monitored in a computer-controlled spectrofluorometer (��excitation = 493 nm, ��emission = 530 nm; QM-8/2003, Photon Technology International, London, ON, Canada). Fluorescence was rapidly quenched by addition of acid-loaded BBMV (a volume containing 150 ��g protein) in the absence of Na+ and under constant stirring. Addition of extravesicular Na+ (Na+-gluconate to a final concentration of 30 mM) activated Na+/H+ exchange and resulted in a proportional increase in fluorescence. Replacement of Na+-gluconate with NMDG-gluconate was used to monitor Na+-independent quenching. Na+/H+ exchange activity was estimated as the initial rate of increase in fluorescence on addition of Na+-gluconate, from which the fluorescence increase with NMDG-gluconate (typically <1/10th of that with Na+-gluconate) was subtracted.

Quantitative comparisons were made between BBMV prepared at the same time from Anacetrapib rats killed on the same day. NHE3 antigen in BBMV. Rat kidney cortex BBMV prepared as above were homogenized in RIPA buffer (150 mM NaCl, 50 mM Tris?HCl, pH 7.4, 5 mM EDTA, 1% Triton X-100, 0.5% deoxycholate, and 0.1% SDS) containing fresh protease inhibitors and cleared by centrifugation (14,000 g, 4��C, 30 min), and protein content was determined by the method of Bradford.