9140 odyssey clx infrared imaging system

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).

Fibroblasts, were grown to confluence in a 15-cm dish, washed twice with phosphate buffered saline, and collected in 600μL RIPA buffer containing Protease inhibitor cocktail (Roche, Manheim, Germany). Samples were sonicated for 10 min in an ultrasonic bath. 25 μg of total protein, as determined by the DC Protein assay (BioRad, Hercules, VA), was loaded on a 4% stacking – 12–15% resolving polyacrylamide gel for immunoblotting with PIGT, PIGS, PIGK, PIGU and GPAA1antibodies. Proteins were transferred to 0.45-μm Immobilon-FL PVDF transfer membranes (Millipore, Billerica, MA) and probed with anti-PIGT (ab80888), anti-PIGU [EPR16424]-C-terminal (ab192255) produced in rabbit (Abcam Inc., Cambridge, MA), anti-PIGS (sc-373701), anti-PIGK (sc-398611), or anti-GPAA1 (sc-373710) produced in mouse (Santa Cruz Biotechnology, Santa Cruz, CA) and monoclonal anti-GAPDH antibody produced in mouse, (G8795, Sigma-Aldrich, St. Louis, MO) or anti-GAPDH produced in rabbit (NB300–327, Novus Biologicals, Littleton, CO). Li-cor Donkey anti-Rabbit IRDye 680RD (Li-cor Inc., Lincoln, NE) and Li-cor Donkey anti-Mouse IRDye 800CW (Li-cor Inc.) were used as secondary antibodies and fluorescence was measured on a 9140 Odyssey CLx Infrared Imaging system (Li-cor Inc.). Band density was analyzed using Image studio Software (Li-cor Inc.).