Western blot analysis

Mouse cerebellum tissues were dissolved in lysis buffer (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% NP-40) with protease inhibitor cocktail (#539134, 1:100 dilution, Calbiochem, CA, USA) for 1 h at 4 °C. After centrifugation (12,000×g × 10 min), supernatants were mixed with an equal volume of sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, 2.5% (v/v) 2-mercaptoethanol, 5% (v/v) glycerol, and 0.0025% (w/v) bromophenol blue). The BCA method (Pierce BCA Protein Assay Kit; Thermo Scientific, IL, USA) was used to determine protein concentrations, which were subsequently equalized across samples. Samples were separated by SDS-PAGE, transferred onto polyvinylidene difluoride membrane Immobilon-P (Millipore) by the semi-dry method, blocked with 5% milk in TBST (10 mM Tris/HCl (pH 8.0), 150 mM NaCl, 0.05% Tween-20), and reacted with the following primary and secondary antibodies diluted in TBST with 0.1% skim milk or Can Get Signal solution (Toyobo, Osaka, Japan): rabbit anti-YAPdeltaC¹³, 1:20,000 (raised against the common COOH-terminal peptide [SVFSRDDSGIEDNDNQ]); rabbit anti-FLAG, 1:2000 (F7425, SIGMA, IL, USA); mouse anti-c-Myc, 1:2000, (#sc-40 Santa Cruz Biotechnology, Dallas, TX, USA); rabbit anti-RORα, 1:1000 (#sc-28612, Santa Cruz Biotechnology); mouse anti-RGS-His, 1:5000 (#34650, Qiagen, Hilden, Germany); rabbit anti-GST, 1:3000 (sc-469, Santa Cruz Biotechnology); rabbit anti-YAP, 1:5000 (#14074S, Cell Signaling Technology, MA, USA); rabbit anti-Tip60, 1:5000 (#PA5-23290, Thermo Scientific); anti-Atxn1, 1:2000 (#MABN37, Millipore, MA, USA); mouse anti-RpA1, 1:1000 (H-7, Santa Cruz Biotechnology), anti-γH2AX, 1:1000 (Ser139, #05-636, Millipore); anti-53BP1, 1:10,000 (NB100-304, Novus Biologicals, CO, USA): anti-GAPDH, 1:5000 (MAB374, Millipore, MA, USA); anti-α-tubulin, 1:3000 (T6199, Millipore); anti-β-actin, 1:1000 (sc-47778, Santa Cruz Biotechnology); HRP-linked anti-rabbit IgG, 1:3000 (NA934, GE Healthcare, IL, USA); HRP-linked anti-mouse IgG, 1:3000 (NA931, GE Healthcare). Primary and secondary antibodies were incubated overnight at 4 °C and for 1 h at room temperature (RT), respectively. ECL Prime Western Blotting Detection Reagent (RPN2232, GE Healthcare) and a luminescent image analyzer (ImageQuant LAS 500, GE Healthcare) were used to detect proteins. Uncropped western blots are shown in Supplementary Fig. ⁹.