Irdye 800cw coupled secondary antibodies

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.

Whole cell lysates were extracted from cells in 2x sample buffer and separated on 10% SDS–PAGE gel and transferred to nitrocellulose membranes (GE Healthcare). The membranes were blocked with Odyssey Blocking Buffer in PBS (Licor), followed by incubation with primary antibodies against human FOXR1 (Biorbyt, rabbit 1:200), GFP (synaptic systems, mouse 1:1000), GAPDH (EMD Millipore, mouse 1:5000), HSPA1A and HSPA6 (Enzo life sciences, mouse 1:1000), DHRS2 (Abcam, rabbit 1:500), Histone H3 (Cell-Signaling, rabbit 1:1000) overnight at 4°C. Proteins recognized by the antibodies were detected with an Odyssey infrared imaging system (LI-COR) using IRDye680RD- or IRDye800CW-coupled secondary antibodies (LI-COR, 1: 20,000).