Gpr32

After appropriate treatments and rinsing with cold phosphate-buffered saline, REC, rMC-1 and PMN were collected in lysis buffer containing protease and phosphatase inhibitors and scraped into tubes. Retinal extracts were prepared by sonication. Equal amounts of protein from the cell or tissue extracts were separated on pre-cast tris-glycine gels (Invitrogen, Carlsbad, CA), and then blotted onto nitrocellulose membranes. After blocking in TBST (10mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (w/v) BSA, membranes were treated with the following primary antibodies: 5-LOX, 15-LOX-1, 15-LOX-2, ALX/FPR2, GPR32 (Abcam, San Francisco, CA) and β-actin (Santa Cruz, Santa Cruz, CA), followed by incubation with appropriated secondary antibodies (Fisher Scientific, Pittsburgh, PA) labeled with horseradish peroxidase. Antigen-antibody complexes were detected using a chemilluminescent reagent kit (Thermo Scientific, Pittsburgh, PA). Western blot images were collected on an Azure Biosystem C500 machine (Azure Biosystems, Dublin, CA) and densitometric analysis was performed.