Sc 32886

HOBs were lysed in freshly prepared ice-cold RIPA buffer. 35 µg total protein, quantified by micro Lowry, was separated by SDS page and transferred to nitrocellulose membranes. Membranes were blocked with 5% BSA in TBS-T for 1 h at ambient temperature followed by overnight incubation at + 4 °C with primary antibodies (sc-32886, sc-30147, sc-50508 and sc-30080 Santa Cruz Biotechnology, Heidelberg, Germany) diluted 1:1,000 in TBS-T. The next day membranes were incubated with the corresponding peroxidase-labeled secondary antibodies (1:10,000 in TBS-T) for 2 h at ambient temperature. GAPDH was used as a loading control. For signal development membranes were incubated for 1 min with ECL substrate solution. Chemiluminescent signals, detected by a CCD camera (INTAS, Göttingen, Germany), were quantified using the ImageJ software.

Total liver protein lysates were obtained and separated in 10% sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE). Gels were then blotted onto PVDF Amersham Hybond-P membranes (GE Health-care, Buckinghamshire, UK) and incubated with their corresponding antibodies (A2228, A9917, C0979 and S2147 from Sigma–Aldrich, St. Louis, Missouri, USA; Ab59546 from Abcam, Cambridge, UK; sc-2004, sc-2490 and sc-32886 from Santa Cruz Biotechnology, Dallas, Texas, USA). β-actin was used as loading control. Blots were developed by enhanced chemiluminescence using an Amersham ECL Plus Western Blotting Detection Reagent (GE Health-care, Buckinghamshire, UK) according to manufacturer’s instructions.

The hippocampus was homogenized in 0.05 M sodium phosphate buffer (pH 6.65). Subsequently, the protein concentration was determined using BCA method (Thermo Scientific Pierce, USA), described by Stich [30 (link)]. CuZn SOD (SOD1), Mn SOD (SOD2), CAT, GPx, GR, BDNF, TH, DAT, and COMT proteins were assayed by Western blot analysis as described previously by Gavrilović et al. [27 (link)]. Antibodies used for the quantification of specific proteins were as follows: SOD1 (SOD-101, Stressgen, USA), SOD2 (SOD-110, Stressgen, USA), CAT (Calbiochem, Germany), GPx (sc-30147 Santa Cruz Biotechnology, USA), GR (sc-32886, Santa Cruz Biotechnology, USA), BDNF (ab6201, Abcam, USA), TH (ab51191, Abcam, USA), DAT (ab18548, Abcam, USA), and β-actin (ab8227, Abcam, USA). After washing, the membranes were incubated in the secondary anti-rabbit (dilution 1 : 5000, Amersham ECL™ Western Blotting Analysis System, UK) antibodies conjugated to horseradish peroxidase. A secondary antibody was then visualized by the Western blotting-enhanced chemiluminescent detection system (ECL, Amersham Biosciences, UK). The membranes were exposed to ECL film (Amersham Biosciences, UK). The result was expressed in arbitrary units normalized in relation to β-actin, which is in accordance with our previous protocol [27 (link)].