Levels of NO were expressed as amplitude selleck compound of signal in unit per weight of dried sample (Amplitude/Wd).Superoxide anion (O2-) spin-trappingAorta and heart samples were allowed to equilibrate in deferoxamine-chelated Krebs-Hepes solution containing 1 hydroxy-3methoxycarbonyl 2,2,5,5-tetramethylpyrrolidin (CMH, Noxygen, Germany) (500 ��M), deferoxamine (25 ��M) and DETC (5 ��M) under constant temperature (37��C) for one hour. The reaction was stopped by placing the samples in ice, subsequently frozen in liquid N2 and analyzed in a Dewar flask by EPR spectroscopy (Magnettech, MS200, Berlin, Germany).. The instrument settings were as follows: temperature, 77�� K; microwave power, 1 mW; amplitude modulation, 0.5 mT; sweep time, 60 s; field sweep, 60 G. Values were expressed in signal amplitude/mg weight of dried tissue (Amplitude/Wd).
Western blottingAorta and heart samples were homogenized in lysis buffer (0.5 M Tris-HCl, 1.86 g/ml EDTA, 1 M NaCl, 0.001 g/ml Digitonin, 4 U/ml Aprotinin, 2 ��M Leupeptin, 100 ��M phenylmethylsulfonyl fluoride (PMSF)). Proteins (20 ��g) were separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes. Blots were probed by an over-night incubation (4��C) with a mouse anti-inducible NOS (iNOS) antibody (BD Biosciences, San Jose, CA, USA), a polyclonal rabbit nuclear factor NF-kB p65 antibody (Abcam, Cambridge, UK), a mouse anti-human phosphorylated (ser32/36)-IkB alpha (P-IkBa) antibody (US Biologica, Swampscott, Massachusetts, USA), an anti-rat I-CAM/CD54 antibody (R&D Systems), a goat COX-1(M-20) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), a goat COX-2 antibody (Santa Cruz Biotechnology), a rabbit polyclonal nuclear respiratory factor Nrf2 (C-20) antibody (Santa Cruz Biotechnology), a rabbit anti-heme-oxygenase-1 (HO-1) polyclonal antibody (Stressgen Bioreagents, San Diego California, USA) or a rabbit anti-heme-oxygenase-2 (HO-2) polyclonal antibody (Stressgen Bioreagents, San Diego California, USA).
Membranes were washed and incubated for one hour at room temperature with a secondary anti-mouse, anti-rabbit or anti-goat peroxidase-conjugated IgG (Promega, Madison, WI, USA).Blots were visualized using an enhanced chemiluminescence system (ECL Plus; Amersham, Buckinghamshire, UK), after which the membranes were probed again with a polyclonal rabbit anti-��-actin antibody (Sigma-Aldrich, Saint Quentin Fallavier, France) for densitometric quantification and normalization to ��-actin expression.Data analysisFor repeated measurements, one-way analysis of variance was used to evaluate within-group differences. Difference between groups was tested using a two-way analysis of variance (repeated time measurements Dacomitinib and treatments as independent variables).