Pierce stripping buffer

Nuclear extracts were isolated from N27 cells treated with 1mM valproate using NE-PER™ Kit (Thermo Scientific) as per manufacturer instructions. Protein homogenates were supplemented with 0.1% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Protein concentrations were determined using the Pierce ™ bicinchoninic acid (BCA) assay kit (Thermo Scientific) and 50 μg of protein sample was loaded per lane on a 4–12% Bis-Tris Polyacrylamide Gel (Invitrogen). Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and blocked in 7.5% non-fat milk in 0.1% Tween 20 and Tris buffered saline (TTBS) for 1 hour at room temperature. The membranes were incubated at 4°C overnight with rabbit anti-H3Ac primary antibody (1:2000; cat# 06-599 Millipore), followed by a 1 hour room temperature incubation with anti-rabbit HRP-conjugated secondary antibody. The bound antibody was detected by SuperSignal^® West Dura Extended Duration Substrate Kit (Thermo Scientific) and imaged using Alpha Innotech Fluorochem imaging system. The membrane was stripped using Pierce Stripping Buffer (Thermo Scientific) and re-probed with rabbit anti-Histone 3 antibody to confirm equal protein loading.

Following 24 h treatment with inhibitors, SK-N-AS cells were lysed with commercial cell lysis buffer (BioVision, Miltipas, CA) containing 0.1 % protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Protein concentrations were determined using the Pierce TM bicinchoninic acid (BCA) assay kit (Thermo Scientific) and 50 μg of protein sample was loaded per lane on a 4–12 % Bis–Tris Polyacrylamide Gel (Invitrogen). Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and blocked in 7.5 % non-fat milk in 0.1 % Tween 20 and Tris buffered saline (TTBS) for 1 h at room temperature. The membranes were incubated at 4 °C overnight with anti-DAT primary antibody (1:2000; cat #MAB369; Millipore), followed by a 1 h incubation with HRP-conjugated anti-rat secondary antibody (MP Biomedicals)at room temperature. The bound antibody was detected by SuperSignal^® West Dura Extended Duration Substrate Kit (Thermo Scientific) and imaged using Alpha Innotech Fluorochem imaging system. The membrane was stripped using Pierce Stripping Buffer (Thermo Scientific, Waltham, MA) and re-probed with anti-tubulin (1:6000; Sigma) antibody to confirm equal protein loading.