Complete and phosphostop

For the determination of oxidative stress parameters and antioxidant components in the brain, frozen tissues were homogenized in RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with Complete and Phospho STOP (Roche, Basel, Switzerland) protease and phosphatase inhibitors as described [20 (link)]. Lipid peroxidation was determined using a commercial TBARS assay kit (CA995, Canvax, Cordoba, Spain). The final malondialdehyde products were detected by fluorescence spectroscopy with excitation/emission at 530 nm/590 nm in a microplate reader (POLARstar Omega, BMG Labtech, Ortenberg, Baden-Wuerttemberg, Germany). Levels of superoxide dismutase (SOD) activity were determined using an SOD assay kit (CA061, Canvax, Córdoba, Spain), according to the manufacturer’s protocol. Absorbance at 450 nm was measured using a POLARstar Omega plate reader. Catalase activities were determined using a commercial Catalase Activity assay kit (CA063, Canvax, Córdoba, Spain) following manufacturer’s instructions. Enzyme activity was detected by fluorescence spectroscopy with excitation/emission at 530 nm/590 nm in a microplate reader (POLARstar Omega).

For the determination of oxidative stress parameters and antioxidant components in the brain, frozen tissues were homogenized in radioimmunoprecipitation assay (RIPA) buffer (Thermo Scientific, Waltham, MA, USA) supplemented with complete and phospho STOP (Roche, Basel, Switzerland) protease inhibitors. Lipid peroxidation was determined using a commercial TBARS assay kit (CA995, Canvax, Córdoba, Spain). The final malondialdehyde (MDA) products were detected by fluorescence spectroscopy, with excitation/emission at 530/590 nm in a microplate reader (POLARstar Omega). Levels of superoxide dismutase (SOD) activity were determined using an SOD assay kit (CA061, Canvax), according to the manufacturer’s protocols. Absorbance at 450 nm was measured using a POLARstar Omega plate reader. Catalase activities were determined using a commercial catalase activity assay kit (CA063, Canvax) following the manufacturer’s instructions. Enzyme activity was detected by fluorescence spectroscopy, with excitation/emission at 530/590 nm in a microplate reader (POLARstar Omega).

Cells were seeded 2 × 10⁵ per well or 2 × 10⁴ per well in 6-well plates. After 24 h recovery, cells were treated for 24 h or 7 days, respectively, with the indicated concentrations of the compounds. Cells were scratched into the medium and after washing with PBS, lysed in lysis buffer (50 mM Tris, 300 mM NaCl, 0.5% Triton-X-100) containing a phosphorylation inhibitor cocktail (Complete and Phospho-Stop, Roche, Austria) for 45 min on ice. After 5 min ultrasound bath, lysates were centrifuged for 15 min at 14 000 rpm at 4 °C. Protein concentration of supernatant was quantified using Micro BCA Protein Assay (Pierce, Thermo Fisher, Austria). 15 μg of proteins were separated by SDS-PAGE (10% gels), and transferred onto a polyvinylidene difluoride membrane for western blotting as described previously.^{51 (link)} The following primary antibodies were used: anti-pH2AX monoclonal rabbit (Cell Signaling, #2577) and anti-β-actin monoclonal mouse (Sigma Aldrich, #A5441). Secondary, horseradish peroxidase-labeled antibodies were anti-rabbit monoclonal mouse (sc-2357, Santa Cruz Biotechnology, Austria) and anti-mouse polyclonal goat (Merck, #A0168) were used in working dilutions of 1 : 10 000.

Cells were lysed in 20 μl of ice-cold lysis buffer A (50 mM Tris, pH 8.0, 100 mM NaCl, 0.5 mM TCEP, 2 mM MgCl2, supplemented with 1x Complete and PhosphoSTOP (Roche)). Lysis was performed by mechanical force using a pestle, pipetting and three cycles of freeze and thaw. After the addition of 20 µl lysis buffer B (lysis buffer A containing 40 mM CHAPS) and 1 μl Benzoase (Novagen), samples were incubated for 1 h on ice and subsequently centrifuged for 10 min at 4 °C and 13,000 r.p.m. to remove cell debris. 20 μl of supernatant was supplemented with 10 μl of 3x Native PAGE sample buffer (60% glycerol v/v, 15 mM Coomassie G250) and separated on BN-PAGE Novex 3–12% Bis-Tris protein gel system (Life Technologies) according to the manufacturer’s instructions. The cathode buffer was supplemented with 0.002% Coomassie G250, and the separation was performed at 4 °C for 60 min at 150 V, then 60 min at 250 V.

For the determination of the oxidative stress parameters and antioxidant components in the brain, frozen tissues were homogenized (RIPA buffer, Thermo Scientific) supplemented with Complete and Phospho STOP (Roche, Basel, Switzerland) protease and phosphatase inhibitors), as described [18 (link)]. The activities of superoxide dismutase (SOD) and catalase were studied with commercially available kits (CA061 and CA063, respectively, Canvax, Córdoba, Spain). Lipid peroxidation was determined using a commercial TBARS assay kit (CA995, Canvax). The final products were detected with a microplate reader (POLARstar Omega, BMG Labtech, Ortenberg, Germany) at 530/590 nm (for catalase and TBARS) or 450 nm for SOD.