Mouse monoclonal anti β actin a1978

Confluent endothelial cells (6-well plates) were washed with sterile PBS and 200 μL RIPA buffer was added (1% Nonidet P40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM NaF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 50 mM Tris-HCl, pH 7.4; Sigma-Aldrich, St. Louis, MO, USA). Cell lysates were centrifuged (8,100×g, 10 min, 4°C), and the protein content was determined by the Lowry method. The proteins (50 µg) were loaded into 12% SDS-PAGE gels and subjected to electrophoresis at a fixed voltage of 100 V. The proteins were transferred to a nitrocellulose membrane (Millipore, USA, 100 V) and then incubated for 1 h in Tris-buffered saline (TBS) with 5% nonfat milk. Next, the membranes were washed three times for 5 min with 1× TBS-Tween solution and incubated overnight with 1:200 rabbit anti-P2Y₂ receptor antibody (APR-010; Alomone Labs, Israel) or 1:5,000 mouse monoclonal anti-β-actin (A1978; Sigma-Aldrich, USA) diluted in 5% BSA 1× TBS-Tween solution. After three washes with 1× TBS-Tween solution for 5 min, the membranes were incubated with HRP-conjugated antirabbit (1:2,000) or antimouse (1:20,000) secondary antibody for 2 h. The image was obtained with the chemiluminescent detection reagent (ECL, Sigma-Aldrich, USA) in the ImageQuant equipment (GE Healthcare, USA).