To analyze protein expression, western blot analysis was performed. Proteins were extracted with RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.1% SDS, 150 mM NaCl, and 2 mM EDTA + protease inhibitors) as follows: Cells were lysed and maintained in ice for 30 min, with vortexing every 10 min. The cells were then centrifuged (Eppendorf® Microcentrifuge 5415; Merck KGaA) at 16,000 × g for 20 min at 4°C. Supernatants were collected and protein concentrations were measured using a Bradford assay (Merck KGaA). Protein (30 μg) was treated with LDS Sample Buffer (Thermo Fisher Scientific, Inc.) and a Sample Reducing Agent (Thermo Fisher Scientific, Inc.). The tubes were then boiled at 100°C for 5 min. Finally, equal amounts of protein (30 μg) were resolved on precast 4-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bolt 4-12% Bis-Tris Plus; Thermo Fisher Scientific, Inc.) in MES SDS running buffer (Thermo Fisher Scientific, Inc.) at 100 V and 35 mA, before entering the gel, and at 165 V and 60 mA after entering the gel. A protein ladder (SeeBlue™ Plus2 Prestained Standard; Thermo Fisher Scientific, Inc.) was used as a reference for protein size. Following running, proteins were transferred to a nitrocellulose membrane (NC; Amersham™ Protran™ Nitrocellulose Blotting Membrane; GE Healthcare) using a semi-dry method in transfer buffer (Tris 25 mM, 0.2 M glycine, 20% methanol). The transfer was performed at 30 V and an amperage calculated based on the membrane's measurements (w × h × 0.8). The protein transfer was verified by immersing the membrane in red Ponceau [Ponceau S solution for electrophoresis (0.2%); SERVA Electrophoresis GmbH] and then washed 3 times for 5 min with PBS-Tween-20 (PBST) 0.1%. Subsequently, the membrane was blocked with 5% milk-PBST (non-fat dried milk; Euroclone S.p.A.) for 1 h at room temperature and then incubated overnight at 4°C with pertinent primary antibodies: Rabbit polyclonal anti-human anti-Gapdh antibody (dilution, 1:1,000; cat. no. A300-639A-M; Bethyl Laboratories Inc.), rabbit polyclonal anti-human anti-caspase-9 (Casp-9; dilution, 1:1,000; cat. no. 9502; Cell Signaling Technology, Inc.), mouse monoclonal anti-human anti-caspase-8 (Casp-8; dilution, 1:1,000; cat. no. 9746; Cell Signaling Technology, Inc.), mouse monoclonal anti-human anti-caspase-3 (Casp-3; dilution, 1:500; cat. no. sc-7272; Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-human anti-BH3 interacting domain death agonist (Bid; dilution, 1:1,000; cat. no. 2002; Cell Signaling Technology, Inc.), rabbit polyclonal anti-human anti-Bcl-2-associated X protein (Bax; dilution, 1:1,000; cat. no. 2772; Cell Signaling Technology, Inc.), mouse monoclonal anti-human anti-cytochrome c (Cyt c; dilution, 1:1,000; cat. no. sc-13156; Santa Cruz Biotechnology, Inc.), rabbit monoclonal anti-human anti-p53 (dilution, 1:1,000; cat. no. 2527; Cell Signaling Technology, Inc.) and rabbit polyclonal anti-human anti-apoptosis independent factor (Aif; dilution, 1:1,000; cat. no. 4642; Cell Signaling Technology, Inc.). The following day, the NC membrane was washed 3 times with PBST 0.1% and incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 3 h: Goat anti-rabbit IgG (H+I) peroxidase/HRP-conjugated (dilution, 1:3,000; cat. no. E-AB-K1813; Elabscience Biotechnology, Inc.) or goat anti-mouse IgG (H+I) FITC conjugated (dilution, 1:10,000; cat. no. A90-116F; Bethyl Laboratories, Inc.). Finally, another 3 5-min washes with PBST were performed prior to membrane development. For signal chemiluminescent detection, the membrane was incubated for 2 min in the dark at room temperature with SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Inc.) and then developed in Alliance Mini (UVITEC Cambridge) equipped with NineAlliance Software (UVITEC Cambridge). Band quantification was carried out using the same software. Membrane stripping was performed prior to the addition of Gapdh where similar molecular weight proteins were previously assessed.

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