Frataxin

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.

Cells were homogenized and lysed on ice with RIPA buffer, including DTT and protease inhibitors. For Western blotting analysis, 40 μg of proteins was subjected to SDS PAGE on 4–12% denaturing gel and probed with the following antibodies: NRF2 (1:500, Abcam, Cambridge, UK), NQO1 (1:1000, Novus Biologicals, Minneapolis, MN, USA), SOD 1/2 (1:5000, Stressgen, Victoria, BC, Canada), Frataxin (1:500, Santa Cruz Biotechnology, Dallas, TX, USA) and GAPDH (1:15,000, Sigma Aldrich, St. Louis, MO, USA) as loading control. Immunoreactive bands were detected using the Lite Ablot Extend Long Lasting Chemiluminescent substrate (Euroclone, MI, Italy). Signals derived from appropriate HRP-conjugated secondary antibodies (Bethyl Laboratories, Montgomery, TX, USA) were captured by Chemi DocTM XRS 2015 (Bio-Rad Laboratories, Hercules, CA, USA) and densitometric analysis was performed using Image Lab software (Version 5.2.1; © Bio-Rad Laboratories).

Fibroblasts (1 × 10⁶) were lysed on ice with RIPA buffer, including DTT and protease inhibitors. 40 µg proteins were subjected to SDS PAGE on 4–12% denaturing gel and probed with the following antibodies: Nrf2 (1:500, Abcam, Cambridge, UK), Nqo1 (1:7000, Novus Biologicals, Minneapolis, MN, USA), Ho-1 (1:3000, Abcam), Frataxin (1:500, Santa Cruz Biotechnology, Dallas, TX, USA), and GAPDH (1:10,000, Sigma Aldrich) as loading control. Immunoreactive bands were detected using the Lite Ablot Extend Long Lasting Chemiluminescent substrate (Euroclone, Milan, Italy). Signals derived from appropriate HRP-conjugated secondary antibodies (Bethyl Laboratories, Montgomery, TX, USA) were captured by Chemi DocTM XRS 2015 (Bio-Rad Laboratories, Hercules, CA, USA) and densitometric analysis was performed using Image Lab software (Version 5.2.1, Bio-Rad Laboratories)