Cocktails of proteases and phosphatases inhibitors

Neuroblastoma SH-SY5Y cells were purchased from ATCC (ATCC CRL-2266) and maintained in DMEM medium supplemented with 10% fetal bovine serum. The cells were cultured in a humidified chamber at 37 °C with 5% CO₂. Cells were plated the day before treatment so that the density of cell culture could reach ~70% confluence. Hydralazine was diluted in culture medium from a stock solution. The final concentration of hydralazine and the duration of the treatment were indicated in the text and the Figure legends.
Oxidative stress was induced in cells with different concentrations of stressors (e.g., hydrogen peroxide or rotenone) to test the efficacy of hydralazine. Hydrogen peroxide treatment was done in 5% serum containing medium. At the end of the treatment, cells were collected and washed once in ice-cold PBS buffer, followed by lysis with RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.2) supplemented with cocktails of proteases and phosphatases inhibitors (Thermo Fisher, Waltham, MA) on ice for 1 h with occasional stirring. The cell lysates were then centrifuged at 10,000 × g for 15 min at 4 °C. Supernatants were collected and protein concentration was measured using the bicinchoninic acid (BCA) assay (Pierce, 23228).

The harvested lysates of cells were prepared by solubilization in RIPA buffer, containing cocktails of proteases and phosphatases inhibitors (Thermo Fisher Scientific, Massachusetts, Waltham, MA, USA). Protein concentrations were determined using the Lowry protein assay [65 (link)]. SDS-PAGE was performed in accordance with the Laemmli method [66 (link)], with 6% stacking gel and 10% running gel. Proteins were transferred from gel to nitrocellulose membrane (BioRad, California, Hercules, CA, USA), followed by blocking with 5% fat-free milk (Valio, Helsinki, Finland) in PBST 1X (PBS supplemented with Tween 20 0.1%) for 1 h at room temperature. Akt, CREB, and their phosphorylated forms were detected by overnight incubation with the corresponding antibodies (#9272S, #4060S, #4820S, #9198S, #9252S, #4668S, #4695S #4377S, Cell Signaling, Massachusetts, Danvers, MA, USA) at 4 °C in 5% milk powder in PBST. These samples were then incubated with HRP-conjugated secondary antibodies monoclonal anti-rabbit IgG (a1949, Sigma-Aldrich, Missouri, St. Louis, MO, USA) for 1 h at room temperature in 5% milk powder in PBST. GAPDH (#2118S, Cell Signaling, Massachusetts, Danvers, MA, USA) was chosen as the house-keeping protein. Antigen-antibody complexes were visualized using an ECL kit and ChemiDoc XRS+ Molecular Imager (BioRad, California, Hercules, CA, USA).