Ab172684

Cardiac tissues were isolated from mice and stimulated with the indicated cardioplegia solutions for 1 hr. Lysates of cardiac tissue homogenates were obtained with lysis buffer (containing (mM) 150 NaCl, 20 Tris, 2 EDTA, 1% Triton X-100, and a protease inhibitor mixture) and were treated as previously described [18 (link)]. 30 μg denatured protein samples was subjected to SDS-PAGE. Proteins were visualized with NKCC1 (ab59191, Abcam), phosphoNKCC1 (#ABS1004, Millipore), Slc26a6 (ab172684, Abcam), CA IV (sc-74527, Santa Cruz Biotechnology), GAPDH (MA5-15738, Thermo Fisher), NHE1 (NBP1-76847, Novusbio), and β-actin (A3854, Sigma) antibodies using the enhanced luminescence (ECL) solution (Thermo Scientific). The intensity of protein band was normalized with that of β-actin as a protein loading control.

HEK293T cells were transfected with the indicated plasmids and after 2 days, lysates were prepared [lysis buffer contained phosphate-buffered saline (PBS), 10 mM Na⁺-pyrophosphate, 50 mM NaF, and pH was adjusted to 7.4. 1 mM Na⁺-orthovanadate, 1% Triton X-100, and a cocktail of protease inhibitors (Roche) were freshly added before each use]. The cells were placed on ice and scraped after addition of lysis buffer. Protein extracts were incubated with Protein G Sepharose beads (Sigma-Aldrich, St. Louis, MO) and anti-His antibody (1:100) (Thermo Fisher Scientific, Waltham, MA) overnight at 4°C. The beads-protein complexes were incubated for 4 h at 4°C, centrifuged, and washed with lysis buffer four times. Samples were prepared for running on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by heating (37°C for 30 min) in SDS sample buffer. Subsequently, the samples were transferred to nitrocellulose membranes and incubated overnight at 4°C with anti-SLC26A6 (1:500) (ab 172684, Abcam) and the next day exposed to the appropriate secondary antibody. Signal was developed using enhanced-chemiluminescence (ECL) substrate (CYANAGEN).