Odyssey infrared imager and software

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.

Mouse cerebellar tissues and 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). The supernatant was recovered by centrifugation (16,000g, 15mins). Protein concentration was assayed by the Bradford protein assay system (Bio-rad). Twenty micrograms of lysates were loaded into 4–12% Bis—Tris gels (Invitro-gen). 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: Iba1(Wako), CD11b(Abcam), MUTYH(Abcam), PARP-1(Trevigen), Frataxin (Santa Cruz), Actin(Abcam), AT1(Abcam), Tubulin (Abcam). 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.

Human b-lymphoblast cell pellets were homogenized with a cell lysis buffer containing Protease/Phosphatase Inhibitor Cocktail (Cell Signaling, Danvers, MA) and PMSF (Sigma-Aldrich, St. Louis, MO). Mixtures of 20ug total protein lysate and 50mM DTT were loaded into 4%–12% Bis–Tris gels (Invitrogen, Carlsbad, CA). Electrophoresis was carried out according to the manufacturer’s recommendations. Following electrophoresis, the proteins were transferred to nitrocellulose membranes by the Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, California) per manufacturer’s instruction and blocked with an Odyssey TBST blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1 hour. Membranes were incubated overnight with the following primary antibodies in blocking buffer: β-tubulin (DSHB-E7; DSHB, Iowa), Frataxin (sc-25820 Santa Cruz Biotechnology), NCF2, PDLIM1, SFTPD (ab109523, ab126628, ab15696 respectively, Abcam, Cambridge, MA) and PRDX5 (LF-MA00017 Abfrontier, Seoul, Korea). The membranes were washed three times with TBS-Tween (EDM Millipore, Billerica, MA) then incubated with anti-rabbit IRDye 680CW and anti-mouse IRDye 800CW-coupled secondary antibodies (LI-COR Biosciences, Lincoln, NE) for 1 hour. Fluorescence was visualized with the Odyssey infrared imager and software (LI-COR) according the manufacturer’s instruction.