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. Author manuscript; available in PMC: 2016 Jan 31.
Published in final edited form as: Free Radic Biol Med. 2014 Nov 28;79:56–68. doi: 10.1016/j.freeradbiomed.2014.11.016

Fig. 7. Effect of DTNB (Ellman’s reagent) and GSH on the formation of DMPO protein radical adducts at the 51kDa and 75 kDa subunits of mitochondrial Complex I.

Fig. 7

A–B, Mitochondria (0.83 mg/ml) isolated from mouse heart (wild type) was pre-incubated with DTNB (1 mM) or GSH (1 mM) at room temperature for 10 min. DTNB-treated or GSH-treated mitochondria were subjected to immunospin trapping analysis with anti-DMPO antibody (1:1000) and normalized to the Western signals with Ab51 (1:10,000) and Ab75 (1:5000) as described in the legend of Fig. 6. C, Mitochondria were pre-incubated with DPI (1 mM) at RT for 10 min, and the reaction was initiated with glutamate (70 mM) plus malate (2.5 mM). The reaction mixture was incubated at 37 °C for 5 min. Aliquots (120 µg protein per lane) of mixture were withdrawn for SDS-PAGE, and immunoblotted with anti-DMPO antibody (1:1000, upper panel) and anti-GSH antibody (1:500) with (lower panel) or without (middle panel) dithiothreitol (DTT). The experiment was performed three times (n=3).