G5 6k centrifuge

Another group of adult Wistar rats (n = 7 rats for each experimental condition) were used for coimmunoprecipitation analysis. Tissue was homogenized with immunoprecipitation buffer supplemented with protease inhibitors (Mini Complete; Roche Molecular Biochemical). Ventricular lysates were centrifuged at 1440 ×g for 5 min in a Beckman G5-6K centrifuge. Supernatants (3.5 mL) were removed and applied to 50 μL protein G Sepharose for 3 h at 4 °C. After centrifugation (5 min, 8000 ×g), lysates were incubated with the anti-CAIX monoclonal antibody and 100 μL protein G Sepharose overnight at 4 °C. The resin was washed and resuspended in SDS-PAGE sample buffer. Samples were electrophoresed on 10% acrylamide gels. Immunoblots were probed with an anti-CAIX antibody or anti-NBC1 antibody, previously mentioned at a 1:1000 dilution. Samples were randomly placed in the immunoblot gel to create internal controls for analysis purposes. Images were cropped to facilitate visual comprehension.

Freshly isolated rat ventricles were separated and homogenized with a Brinkmann Homogenizer (Brinkmann Instruments, Westbury, NY, USA) in 4 ml of ice-cold solution, containing 0.32 M sucrose, 1 mM EGTA, 0.1 mM EDTA, 10 mM Hepes, pH 7.5 and protease inhibitors (Complete Mini protease inhibitor cocktail tablets, Roche, Germany). Homogenates were centrifuged at 14409g for 5 min in a Beckman G5-6K centrifuge. Supernatants were removed and centrifuged at 66,7009g for 30 min at 4 °C in a Beckman TLA 100.4 rotor. The resulting membrane fraction was resuspended in 300 ll of PBS containing (mM): NaCl 140, KCl 3, Na 2 HPO 4 6.5, KH 2 PO 4 1.5, and pH 7.5. Protein was quantified by Bradford assay, and 125 lg of protein used for immunoblots.