4.7. Western Blot Analysis

SH-SY5Y cells were seeded and treated for 24 h as described above, and then processed following Ferlazzo and co-workers [40]. Briefly, the cells were washed with ice-cold PBS and lysed in a buffer containing: 0.32 M sucrose, 10 mM Tris-HCl pH 7.4, 1 mM EGTA, 2 mM EDTA, 5 mM NaN3, 10 mM 2-mercaptoethanol, 50 mM NaF, and protease inhibitor (Roche Diagnostics Corporation, Indianapolis, USA). The homogenates were chilled on ice for 20 min, centrifuged at 9600× g at 4 °C for 1 min, and then the supernatant (cytosolic extract) was collected. Following, pellets were suspended in a lysis buffer containing 0.1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EGTA, 1 mM EDTA, and protease inhibitors (Roche Diagnostics Corporation), kept on ice for 30 min and centrifuged at 9600× g at 4 °C for 10 min. The supernatant (nuclear extract) was collected and stored at −80 °C until use. Protein concentrations were determined using a Bio-Rad Protein Assay (Bio-Rad Laboratories) using BSA as the standard. Proteins were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF transfer membrane (Immobilon-P PVDF, Merck Millipore division of Merck KGaA, Darmstadt, Germany), blocked with PBS containing 5% non-fat dried milk for 1 h at room temperature, and subsequently probed with the following antibodies at 4 °C overnight: Cleaved-caspase 3 (1:1000), Cleaved-caspase 9 (1:1000), Bax (1:500), NFκBp65 (1:1000; AbCam), GAPDH (1:1000), Lamin B1 (1:500) (all from Cell Signaling Technology, Inc, Danvers, MA, USA, Cell Signaling Technology), p21 (1:1000; Merck Millipore), and p53 (1:2000; Abcam, Cambridge, UK). Then membranes were incubated with horseradish peroxidise-conjugated goat anti-mouse or anti-rabbit IgG secondary antibodies (1:2000; Santa Cruz Biotechnology, Inc., Dallas, Texas, USA) at room temperature for 1 h. Protein bands were visualized using an enhanced chemiluminescence system (Luminata™ Forte, Western HRP substrate; Millipore), acquired with the ChemiDoc™ MP System (Bio-Rad Laboratories) and quantified with the ImageJ software.