E8655

Western blot analysis was carried out for Ube3a^−/− and Ube3a^+/+ MEFs. The cells were washed with PBS, collected, and homogenized by sonication with an ice-cold lysis buffer containing the following (in mM): 10 HEPES pH 7.5, 150 NaCl, 50 NaF, 1 EDTA, 1 EGTA, 10 Na4P2O7 (EMD Millipore, Billerica, MA, USA), PMSF (Roche, Mannheim, Germany), and protease inhibitor cocktail (Roche, Mannheim, Germany). The samples (15 µg) were loaded on an SDS PAGE 4–20% gradient, followed by a transfer to polyvinylidene difluoride (PVDF; Roche, Mannheim, Germany) membranes and probed with primary antibodies using standard techniques. The primary antibodies and the dilutions for the Western blots were as follows: BAX 1:2000 (α-rabbit; Abcam #ab182733, Cambridge, UK), BCL-2 1:2000 (α-rabbit, Abcam #ab182858, Cambridge, UK), and UBE3A 1:1000 (α-mouse; Sigma-Aldrich #E8655, St. Louis, MO, USA). β-ACTIN 1:40,000 (α-mouse; MP Biomedicals #69100, Irvine, CA, USA) was used as a loading control. Secondary antibodies were used, respectively. The blots were developed and imaged using the Image Quant LAS 4000 system. All signals were normalized by the total protein and quantified using Image Studio Lite Ver 5.2 software.

To collect tissue for Western blot analysis, brain tissue was dissected from adult mice and immediately frozen in liquid nitrogen. The lysates were prepared by homogenization in lysis buffer (10 mM Tris-HCL pH 6.8, 2.5% SDS) supplemented with protease inhibitor cocktail (P8340, Sigma–Aldrich). After centrifugation (6000 rpm for 5 min) supernatants were collected and concentration was measured using the Pierce BCA protein assay kit (#23225, ThermoFisher Scientific). A total of 20 μg of each sample was loaded on the gel and a wet transfer was performed. The blotted nitrocellulose membrane was probed with antibodies directed against E6AP (E8655 Sigma–Aldrich; 1:1,000, RRID: AB_261956) and Actin (MAB1501R Millipore; 1:20,000, RRID: AB_2223041). A fluorophore-conjugated secondary Goat anti-mouse antibody (Westburg, IRDye 800CW; 1:15,000, RRID: AB_2687825) was used and the protein was detected using Li-cor Odyssey Scanner system. Quantification was done using Odyssey 3.0 software (Li-cor Biosciences). Number of animals used for quantification was 4 for each genotype.