Sc 8310

Western blot analysis was performed using the protein extracts from ELISA quantifications. Aliquots of each sample containing equal amounts of protein (30 μg) were subjected to SDS-polyacrylamide gel electrophoresis, and β-actin was used as loading control. Gels were transblotted onto a polyvinylidene difluoride membrane, and the blots were blocked in 3% skim-milk for 1 h at room temperature, followed by incubation overnight at 4 °C with a primary goat polyclonal antibody directed to ICAM-1 (1:200 dilution; sc-1511; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or with primary rabbit polyclonal antibody directed to iNOS (1:200 dilution; sc-8310; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or GFAP (1:200 dilution; sc-9065; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Finally, blots were incubated for one hour at room temperature with HRP-conjugated secondary antibodies (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and developed with Clarity Western enhanced chemiluminescence substrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Images were acquired (ChemiDoc XRS⁺; Bio-Rad Laboratories, Inc., Hercules, CA, USA), and the optical density (OD) of the bands was evaluated (Image Lab 3.0 software, Bio-Rad Laboratories, Inc., Hercules, CA, USA). The data were normalized to the corresponding OD of β-actin. All experiments were performed in duplicate.

Aliquots of protein (20 μg) were separated on 6.0% or 15% SDS-polyacrylamide gels and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Millipore, Tokyo, Japan). The blots were blocked by treatment with 5.0% skimmed milk in Tris-buffered saline containing 0.1% Tween-20, and were then incubated with antibodies. The following antibodies were used: SOD1 [16 (link)], SOD2 [16 (link)], NOS2 (sc-8310, Santa Cruz Biotechnology), cyclooxygenase-2 (COX2) (sc-1746, Santa Cruz Biotechnology), peroxiredoxin (Prx) 1 (Prx1) [19 (link)], Prx2 (LF-PA0091, AbFrontier, Seoul, Korea), Prx3 (LFMA0044, AbFrontier), Prx4 [20 (link)], glutathione peroxidase 1 (Gpx1) [21 (link)] and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sc-25778, Santa Cruz Biotechnology). After incubation with horseradish peroxidase-conjugated anti-mouse (sc-2005, Santa Cruz Biotechnology) or anti-rabbit (sc-2004, Santa Cruz Biotechnology) secondary antibodies, the immunoreactive bands were detected using an Immobilon western chemiluminescent HRP substrate (Millipore) on an image analyzer (ImageQuant LAS500, GE Healthcare, Hino, Japan). The relative amounts of each protein were quantified using the Image J software [22 (link)].