Immobilon fl polyvinylidene fluoride pvdf transfer membrane

Cells were grown to confluence in a 15-cm dish, washed twice with phosphate buffered saline (PBS), and cell lysate was collected in 600 μL radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, St Louis, Missouri, USA) containing Protease inhibitor cocktail (Roche, Manheim, Germany). Samples were sonicated for 10 min in an ultrasonic bath. 25μg of total protein, as determined by the DC Protein assay (BioRad, Hercules, Virginia, USA), was loaded on a 4% stacking—10% resolving polyacrylamide gel. Proteins were transferred to a 0.45-μm Immobilon-FL polyvinylidene fluoride (PVDF) transfer membrane (Millipore, Billerica, Massachusetts, USA) and probed with anti-iPLA2 (C-terminal region) produced in rabbit (SAB4200130, Sigma-Aldrich, St Louis, Missouri, USA) and monoclonal anti-GAPDH antibody produced in mouse (G8795, Sigma-Aldrich, St Louis, Missouri, USA). Li-cor Donkey antirabbit IRDye 680RD (Li-cor, Lincoln, Nebraska, USA) and Li-cor Donkey anti-mouse IRDye 800CW (Li-cor, Lincoln, Nebraska, USA) were used as secondary antibodies and fluorescence was measured on a 9140 Odyssey CLx infrared imaging system (Li-cor, Lincoln, Nebraska, USA). Bands were analysed using Image Studio software (Li-cor, Lincoln, Nebraska, USA).