Mouse cerebellar tissues and B-lymphocyte cell pellets were homogenized with a cell lysis buffer (Cell Signaling) with Halt phosphatase inhibitor (Thermo-Fisher), complete protease inhibitors (Roche) and PSMF (Sigma-Aldrich). Thirty micrograms of lysates were loaded into 4–12% Bis–Tris gels (Invitrogen). Electrophoresis was carried out according to the manufacturer's recommendations. Following electrophoresis, the proteins were transferred to nitrocellulose membranes by the iBlot device (Invitrogen), blocked with an Odyssey blocking buffer (LI-COR Biotechnology) for 1 h. Membranes were incubated overnight with the following primary antibodies in blocking buffer: COX1 (ab133319 for human, ab133319 for mouse; Abcam), COX2 (ab62331 for human, ab6665 for mouse; Abcam), phospho-CREB (4095), phospho-cJun/AP1 (5464), phospho-NFκB (4025; Cell signaling), cPLA2 (sc-454), phospho-cPLA2 (sc-34391; Santa Cruz Biotech, Santa Cruz, CA, USA), β-actin (A5441; Sigma-Aldrich), and β-tubulin (DSHB-E7; DSHB, Iowa). Subsequently, the membranes were incubated with a corresponding pair of IRDye 680CW and IRDye 800CW-coupled secondary antibodies (LI-COR). Proteins were visualized with the Odyssey infrared imager and software (LI-COR) according the manufacturer's instruction.
Hayashi G., Shen Y., Pedersen T.L., Newman J.W., Pook M, & Cortopassi G. (2014). Frataxin deficiency increases cyclooxygenase 2 and prostaglandins in cell and animal models of Friedreich's ataxia. Human Molecular Genetics, 23(25), 6838-6847.
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