15 lox 1

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.

Formalin-fixed paraffin-embedded colon and spleen tissues of mouse (4 μm thick) were stained by hematoxylin-eosin (HE) or were dewaxed for immunohistochemistry. Endogenous peroxidase activity was then inhibited by exposure to 3% hydrogen peroxide for 30 min, and antigen retrieval was realized through boiling for 30 min in citrate buffer (pH 6.0, 10 mM). The sections were then blocked with 5% BSA and incubated with 15-lox-1 (1 : 200; Abcam), F4/80 (1 : 200; Santa Cruz), and PCNA (1 : 200; Cell Signaling Technology) primary antibody overnight at 4°C. Finally, sections were visualized using diaminobenzidine (DAB) substrate and counterstained with hematoxylin for microscopic examination.

The expression of 15‐LOX‐1 and 15‐LOX‐2 was assessed in washed human leukocyte‐depleted platelets (3 × 10⁸ platelets/mL) and non‐depleted platelets (3 × 10⁸ platelets/mL). The purified 15‐LOX‐1 (3.7 μg/lane), 15‐LOX‐2 (7.4 μg/lane), and 12‐LOX (7.5 μg/lane) enzymes were used as control. 5X Laemmli sample buffer was added to the platelets, and samples were boiled and then separated on a SDS‐PAGE gel. Western blots were performed with antibodies to 15‐LOX‐1 (Abcam) or 15‐LOX‐2 (Abcam), and β‐actin (Cell Signaling Technology).