Ab6046

In the first part of the experiment, to assess the expression of Ubc9 proteins, cells seeded in 75 cm³ bottles at a density of 5 × 10⁶ were washed with phosphate-buffered saline (PBS) and resuspended in a Tris buffer (pH 7.0) containing protease inhibitors (Roche, F. Hoffmann-La, Basel, Switzerland). After sonication, 20 μg of the total protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a PVDF membrane. Thereafter, non-specific binding was blocked with 5 % bovine serum albumin in TBST for 1 h at room temperature. Membranes were then incubated overnight at 4 °C with rabbit anti-Ubc9 (1:2000, ab6046; Abcam, Cambridge, UK) and rabbit anti-β-actin (1:4000, ab6046; Abcam), respectively. After rinsing, the membranes were incubated with an anti-rabbit secondary antibody for 2 h at room temperature. Bands were visualized using the SuperSignal West Pico Chemiluminescence Substrate from Pierce (Rockford, IL, USA).
In the second part of the experiment, to examine the expression of Ubc9, the animals were decapitated and the cerebral cortex was rapidly harvested. Subsequent protocols were the same as above.

Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 4% to 20% gradient gel (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and transferred onto polyvinylidene fluoride or nitrocellulose membranes (Thermo Fisher Scientific). Membranes were blocked either with 5% bovine serum albumin (BSA, Sigma-Aldrich) or 5% milk in Tris-buffered saline (TBS). Proteins were analyzed with rabbit antibodies against myelin basic protein (Mbp, 1:400, AB980, Thermo Fisher Scientific), microtubule-associated protein 2 (Map2, 1:500, ab24640, Abcam, Cambridge, UK) and β-Tubulin (1:4000, ab6046, Abcam) in 5% milk (for Mbp) or 5% BSA (for Map2 and β-Tubulin) in TBS-Tween 20 (TBS-T, Sigma-Aldrich) overnight at 4 °C. Horseradish peroxidase-coupled donkey anti-rabbit (1:1000, GE Healthcare Life Science, Piscataway, NJ, USA) antibody was used as a secondary antibody and incubated with the membranes for 1 h at room temperature. Binding was detected using the chemiluminescent Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences) on a Chemidoc XRS+ system (Bio-Rad). Pixel summation of individual bands was performed with ImageJ Software (NIH, Bethesda, MD, USA). The ratio between Mbp/Map2 and β-Tubulin was calculated for each animal.