Irdye 680cw

20 µg of total protein was loaded onto a 10% SDS-PAGE gel (Cat: #1610158, Bio-Rad, USA) and blotted to Nitro cellulose membranes (Cat: 88018, Thermo Fisher Scientific, the Netherlands) followed by incubation with block buffer (Cat: P/N 927-40000, Licor, Bioscience, USA). Protein levels were assessed by immunoblotting using specific antibodies against LAT2 (Cat: 11986S, Cell Signaling Technology, Leiden, the Netherlands, 1:1000), and β-actin (Cat: ab8229, Abcam, Cambridge, UK, 1:500) as a loading control, followed by incubation with secondary antibodies (IRdye 680 CW, Cat: P/N 925-68071, P/N 925-68074; IRDye 800 CW, Cat: P/N 925-32211, P/N 925-32214, Licor Bioscience, USA) and detection of signals using the Odyssey imaging system (Licor Bioscience, USA).

Whole-cell lysates were prepared by sonication in cold RIPA buffer containing 1× protease inhibitor cocktail (Roche), 10 mM β-glycerophosphate, 1 mM sodium orthovanadate, 10 mM sodium fluoride, and 1 mM phenylmetnylsulfonyl fluoride. Equal amounts of protein samples were separated by SDS-PAGE gel electrophoresis and transferred to nitrocellulose membranes, then incubated with the primary antibodies overnight. The β-actin was used as the internal control of protein loading, and the immunoblots were recorded with the Odyssey infrared imaging system (LI-COR) after exposure to appropriate IRDye 680CW or IRDye 800CW secondary antibodies (LI-COR, Lincoln, Nebraska) for 1 h at room temperature.^{49 (link)}

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 solubilised on ice in immunoprecipitation (IP) buffer [25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% (v/v) IGEPAL CA-630] supplemented with 2 mM Na₃VO₄, 50 mM β-glycerophosphate, 50 mM NaF, 10 mM NEM, 1 mM PMSF and protease and phosphatase inhibitor cocktails [all reagents form Sigma, expect for Tris, NaCl and EDTA (Thermo Fisher Scientific), P2 and P3 phosphatase inhibitor (Merck P5726 and P0044) and protease inhibitor (Merck 4693132001)] by shaking at 4°C for 1 h. Detergent-insoluble material was cleared by centrifugation at 17,000 g for 10 min at 4°C. For CIP treatment, post-nuclear lysates prepared as described above were treated with CIP (New England Biolabs; 0.5 U/µl final) for 1 h at 37°C.
To perform western blotting of total cell extracts, cells in dishes were washed three times in PBS, and then lysed in 2× SDS-PAGE sample buffer and boiled (Rabaptin-5 hyperphosphorylation samples were gently heated at 37°C for 1 h). Samples were run on polyacrylamide gels and transferred to PVDF membranes (Millipore). Blots were probed with IRDye 680 CW and IRDye 800 CW secondary antibodies (Li-Cor Bioscience) and scanned with a Li-Cor Bioscience Odyssey scanner. Quantitation was performed using ImageJ software.