*4.7. Protein Preparation and Western Blotting*

The cells (0.5 <sup>×</sup> 106 cells/well) were seeded in 6-well plates and incubated overnight. The medium was replaced with fresh medium (1 mL/well) containing the investigated actinoporin at the indicated concentrations, and the cells were incubated for the next 48 h. Cells were harvested by scrapers, pelleted (centrifugation, 5 min, 453× *g*), and washed with PBS (10 mL/samples followed by centrifugation, 3 times). Cells were lysed with 70 μL/sample of lysis buffer (0.88% (*w*/*v*) NaCl, 50 mM Tris-HCl (pH 7.6), 1% NP-40 (*v*/*v*), 0.25% (*w*/*v*) sodium cholate, 1 mM PMSF, 1 mM Na3VO4, containing cOmplete™ EASYpacks protease inhibitors cocktail and PhosSTOP™ EASYpacks phosphatase inhibitors cocktail (Roche, Mannheim, Germany)) on ice for 20 min. Lysates were frozen for 1 h, centrifuged (10 min, 11,170× *g*), the protein-containing supernatants were taken, and the protein concentrations were evaluated using the Bradford assay. Protein extracts were diluted with loading buffer according to the manufacture's recommendations, heated for 5 min at 99 ◦C, and subjected to electrophoresis in 4–12.5% gradient SDS-PAGE (Cat. No. 4568083, Bio-Rad, Hercules, CA, USA) at 120 V. A quantity of 20 μg of total protein was loaded to each slot of the gel. Proteins were then transferred from gel to a 0.2 μm pore PVDF membrane (Millipore, Bedford, MA, USA) using the Bio-Rad transfer system (Cat. No. 10026938, Bio-Rad) and the membranes were blocked with 5% (*w*/*v*) BSA in 0.05% Tween-20/TBS. The membranes were consequently treated with the primary and secondary antibodies, according to the manufacturer's protocol. Signals were detected using the enhanced chemiluminescence system (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer's protocol. The following primary and secondary antibody were used: anti-caspase-3 (mAb, anti-rabbit, #9662, dilution 1:1000, Cell Signaling), anti-MMP-2 (mAb, anti-rabbit, #13132, dilution 1:1000, Cell Signaling), anti-MMP-9 (mAb, anti-rabbit, #13667, dilution 1:1000, Cell Signaling), anti-PARP (pAb, anti-rabbit, #9542, dilution 1:1000, Cell Signaling), anti-Bcl-2 (pAb, anti-rabbit, #2876, dilution 1:1000, Cell Signaling), anti-α-tubulin (mAb, anti-mouse, T5168, dilution 1:5000, Sigma-Aldrich), anti-Bax (mAb, anti-rabbit, #5023, dilution 1:1000, Cell Signaling),anti-β-actin (mAb, anti-mouse, #CP01, dilution 1:10,000, Calbiochem), secondary anti-mouse IgG-HRP (sheep, #NXA931, dilution 1:10,000, GE Healthcare), secondary anti-rabbit IgG-HRP (goat, #7074, dilution 1:5000, Cell Signaling).
