FIG. 4.
Abundance, redox status, and response of Trx system from WT and db/db mitochondria to preincubation with GSHee. Freshly isolated heart mitochondria from WT and db/db mice were handled and analyzed for TrxR2 and Trx2 protein abundance by Western blot (WB) and for Trx-SS and Trx(SH)2 status by redox WB. A: Shown are three representative examples (left) and the statistical comparison between WT and db/db (n = 6 experiments) (right). As in Supplementary Fig. 3, protein was normalized to total protein abundance based on Direct Blue 71 staining. Trx(SH)2 and Trx-SS were determined in fresh, active mitochondria (150–200 μg mitochondrial protein), under baseline (nonenergized) (B), state 4 (St 4) (5 mmol/L G/M), and state 3 (St 3) (+1 mmol/L ADP) of respiration in paired samples as described elsewhere (33). B: Representative redox WB of Trx2 from WT and db/db mitochondria (left), and the amount of Trx(SH)2 (right) (top, in %) and redox potential (bottom, in mV) comparatively between WT and db/db mitochondria, in the absence or presence of GSHee preincubation (3 mmol/L GSHee for 30 min; n = 6 experiments). *P < 0.05; ***P < 0.001.