Anti β actin clone ac 40

Tissues were lysed on ice in radioimmunoprecipitation assay buffer (Tris 50 mM, NaCl 0.15 nM, EDTA 10 mM pH 7.4, β-mercaptoethanol 0.1%, Tween-20 0.1% and anti-protease cocktail 1:100) with protease inhibitors and quantified using the bicinchoninic acid method. Protein lysates (50 µg) were resolved using 10% SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked for 3 h at room temperature with 5% skimmed milk in Tris-buffered saline with Tween-20 prior to immunoblotting overnight at 4°C with anti-P2X₇R (cat. no. ab93354, Abcam, Cambridge, UK; dilution, 1:300) or anti-β actin (clone AC-40; dilution, 1:50,000; Sigma-Aldrich; Merck KGaA), followed by treatment with the respective secondary antibodies 1 h at room temperature, including horseradish peroxidase (HRP)-conjugated horse anti-mouse IgG (cat. no. PI200) and HRP-conjugated goat anti-rabbit IgG (cat. no. PI1000; dilution, 1:1,000; both Vector Laboratories, Inc., Burlingame, CA, USA). Signals were detected using an enhanced chemiluminescence reaction (ProteinSimple; Bio-Techne, Minneapolis, MN, USA). The intensity of bands was quantified using Gel-Pro Image Analysis software version 32 (Media Cybernetics, Inc., Rockville, MD, USA).