*4.5. Immunoblotting Analysis*

The immunoblotting analysis was carried out as described previously [72]. Briefly, the Saos-2 cells (3 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL) were seeded into 6-well plates and, after attachment, cultured in medium with 2% FBS without AKG or with AKG for 72 h. The cells were harvested and lysed for 40 min on ice in RIPA buffer (Sigma-Aldrich) supplemented with protease and phosphatase inhibitor cocktail (Sigma-Aldrich) and centrifuged (10,000× *g* for 10 min at 4 ◦C). The total protein concentrations were determined using a BCA protein assay kit (Pierce® BCA Protein Assay Kit, Thermo Scientific, Rockford, IL, USA). For Western blot analysis, supernatants of RIPA cell lysates were solubilized in 4 x Laemmli sample buffer (Bio-rad Laoratories Inc., Hercules, CA, USA) and denaturized (for 5 min at 100 ◦C). Equal amounts of the protein extracts (40 micrograms) were electrophoresed on SDS-PAGE (Bio-Rad Laoratories Inc., Hercules, CA, USA, Mini-Protean® Tetra Cell) and transferred onto a PVDF membrane (Merck Millipore Corporation, Burlington, MA, USA). After blocking with 5% non-fat dry milk/TBS/0.1% Tween (Sigma-Aldrich) for 1h at RT and washing, the membranes were incubated overnight at 4 ◦C with the following primary antibodies: anti-caspase-8 and anti-caspase-9 (1:400) as well as anti-Bcl-2 and anti-Bax (1:1000) (all antibodies from Santa Cruz Biotechnology Inc. CA, USA). Next, primary antibodies were detected by horseradish peroxidase (HRP)-conjugated rat, mouse, or goat secondary antibodies (1:2000, Santa Cruz Biotechnology Inc. CA, USA), and protein-antibody complexes were visualized with the ECLTM Western Blotting Analysis System (AmershamTM GE Healthcare, Buckinghamshire, UK) and Molecular Imager® ChemiDocTM XRS<sup>+</sup> (Bio-rad Laoratories Inc., Hercules, CA, USA) equipped with ImageLabTM Version 3.0 Software. The blots were reprobed with antibodies against β-actin (1:500, Santa Cruz Biotechnology Inc., CA, USA) used as a load control. Additionally, protein molecular markers (Precision Plus ProteinTM Dual Color Standards, Bio-rad Laoratories Inc., Hercules, CA, USA) were loaded onto electrophoretic gels to control the molecular weight of protein bands. Densitometric measurement of chemiluminescent signals was performed using software ImageLabTM Version 3.0. The optical density of the bands was normalized to β-actin levels.
