Bcl2 associated x (bax)

After two washings with cold PBS, cells were lysed in ice-cold lysis buffer (SDS 1% EDTA 10 mM; Tris-HCl pH8,1 50 mM; protease inhibitors, Na3VO4 1 mM, NaF 100 × ) and extracts were sonicated. Protein extracts were separated by SDS-PAGE, transferred onto a PVDF membrane (Millipore, Billerica, MA, USA) and revealed with a chemiluminescence kit (Millipore). Presented Western-Blot are representative of three independent experiments. Following antibodies were used: actin (MAB1501R, Millipore), β-tubulin (T0198, Sigma), BAX (A3533, Dako), BCL-xL (1018-1, Epitomics), cytochrome c (BD-Pharmingen San Diego, CA, USA), MCL-1 (sc-819, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), NOXA (ALX-804-408, Enzo Life Science, New York, NY, USA), PUMA (12450, Cell Signalling), p21 (2947, Cell Signalling), p53 (#554294, BD-Pharmigen). The above antibodies against BAX, BCL-xL and p53 were also used for immunoprecipitations, as those against BAX 6A7 (ab5714, Abcam, Cambridge, UK) and FLAG-tag (F1804, Sigma).

To assess the ability of resveratrol to induce the apoptotic process, an ICC reaction was performed. The cells of the tested lines were plated on Millicell EZ SLIDES eight-well glass slides (Merck Millipore, Gernsheim, Germany) in the following amounts: EPP85-181P—1 × 10⁴ cells/well, EPP85-181RNOV—1 × 10⁴ cells/well, AsPC-1—1.5 × 10⁴ cells/well and H6c7—1.5 × 10⁴ cells/well. 24 h later, cells were treated with resveratrol at concentrations of 0, 25, 50 and 100 µM for 48 h. After this time, cells were fixed with methanol-acetone (1:1) for 10 min at 4 °C. The ICC reaction was performed on an Autostainer Link48 (Dako, Glostrup, Denmark). The following primary antibodies were used: Bcl-2 (Dako, Glostrup, Denmark), Bax (Santa Cruz Biotechnology, Dallas, TX, USA) and activated Caspase-3 (Cell Signaling Technology, Boston, MA, USA). Slides were first incubated with primary antibodies against Bcl-2 (ready-to-use), Bax (1:25) and activated Caspase-3 (1:400) for 20 min at room temperature, followed by 20 min with EnVision FLEX/HRP (Dako, Glostrup, Denmark). In the next step, the slides were incubated for 10 min with 3,3’-diaminobenzidine (DAB, Dako. Glostrup, Denmark). The slides were counterstained with EnVision FLEX Hematoxylin (Dako, Glostrup, Denmark) and sealed with coverslips in a mounting medium. The ICC reaction was assessed using a BX-41 light microscope (Olympus, Tokyo, Japan).