Western Blot Analysis of Neuronal Proteins

We followed the same procedures of Western blot analysis as previously described [9 (link), 38 (link)]. In brief, 72 h after drug treatment, we collected the cultured neurons from 6-well plates and extracted total protein with protein extraction buffer. Bradford assay was performed to measure protein concentration, and 30 μg of total protein was loaded for Bis-polyacrylamide gel electrophoresis (Bio-Rad). Electrophoresed proteins were transferred to nitrocellulose membrane (0.45 μm, Bio-Rad). The membranes were blocked with 5% non-fat milk in TBS-T at room temperature for 30 min followed by overnight 4 °C incubation with primary antibodies (rabbit anti-GFP, 1:1000, Novus Biologicals; rabbit anti-Ube3a, 1:1000, Bethyl Lab; mouse anti-actin; 1:5000, Sigma). The next day, the membranes were rinsed with TBS-T three times and incubated with HPR-conjugated secondary antibodies for 1 h at room temperature (goat anti-rabbit, 1:1000, Vector Lab or goat anti-mouse, 1:1000, Vector Lab). Following secondary antibody incubation, the membranes were rinsed with TBS-T at room temperature for 1 h (4–5 times) and ECL substrates (Bio-Rad) were used to visualize immunostaining using an Amersham Imager 600 (AI600, GE Life Sciences).