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. 2010 Jul 15;49(2):282–293. doi: 10.1016/j.freeradbiomed.2010.04.018

Fig. 5.

Fig. 5

Structural consequences of perlecan modification by peroxynitrite. Perlecan (330 nM) in 0.1 M phosphate buffer, pH 7 (lane 1) was exposed for 20 min at 22 °C to peroxynitrite at molar ratios of 250 (lane 2), 500 (lane 3), 1000 (lane 4), 1000 in the presence of 25 mM bicarbonate (lane 5), heparinase III for 16 h at 37 °C (lane 6), heparinase III followed by 1000-fold molar excess of peroxynitrite (lane 7), and dONOO (lane 8), prior to separation on 3-8% Tris-acetate gels under reducing conditions for 1 h at 150 V and subsequent western blotting to nitrocellulose. (A) Stains-all/silver stain of gel. (B) Western blot probed for 3-nitroTyr formation with mAb against 3-nitroTyr (HM11). (C) Western blot probed for heparan sulfate epitopes with mAb 10E4. (D) Western blot probed for perlecan domain III with mAb 7B5. Position of molecular mass markers are shown for reference: ◁ perlecan heparan sulfate, ◀ perlecan protein core.