0.45 μm nitrocellulose blotting membranes

Rat aortas were lyzed with RIPA lysis buffer (Amresco, Solon, OH, USA) containing protease inhibitors (Roche, Mannheim, Germany) with subsequent homogenization and centrifugation at 12,000 g at 4 °C for 20 min. Protein content was quantified with bicinchoninic acid protein assay kit (Pierce Biotechnology, Life Technologies). The proteins were separated on 10% SDS gel (30 μg per sample) and transferred onto 0.45 μm nitrocellulose blotting membranes (Amersham, Freiburg, Germany). Membranes were blocked with 5% milk for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies against A1R (1:500, Sigma-Aldrich, SL, USA, product number: A-268), P2X₇R (1:200, Alomone Labs, Israel, product number: APR-004), P2Y₆R (1:200, Alomone Labs, Israel, product number: APR-011) and GAPDH (1:2500, Sigma-Aldrich, MO, USA, product number: G9545), followed by incubation with secondary antibodies for 1 h at room temperature (goat anti-rabbit, 1:20,000, LICOR IR Dye 800CW, product number: 926-32211) [21 (link)]. Band densities were analyzed with Image Studio Lite Version 5.2 (LI-COR Bio-sciences, Bad Homburg, Germany). Data obtained were normalized to GAPDH and displayed with arbitrary units.

Cells were lysed in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 10 mM NaF, and 2 mM Na₃VO₄) containing a protease inhibitor cocktail (1 μg/ml aprotinin, 1 μg/ml antipain, 5 μg/ml leupeptin, 1 μg/ml pepstatin A, and 20 μg/ml PMSF). Cell lysates were clarified by centrifugation at 13,000 rpm for 15 min at 4 °C, denatured with SDS-PAGE sample buffer, and analyzed by SDS-PAGE. Proteins were transferred to 0.45-μm nitrocellulose blotting membranes (Amersham Biosciences, Piscataway, NJ, USA) and probed with the appropriate antibodies. Signals were detected with an Odyssey CLx imager (Lincoln, Nebraska, USA) and analyzed using the Image Studio Lite software (LI-COR Biosciences, Lincoln, NE, USA)^{18 (link),30 (link)}.