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. 2012 Sep 13;287(45):38210–38219. doi: 10.1074/jbc.M112.392225

FIGURE 4.

FIGURE 4.

ATG and ebselen synergically affected the Trx system activity in vitro. Trx system activity was determined using an insulin assay in the presence or absence of ATG and/or ebselen. A, wild-type Trx1. B, C62S/C73S Trx1. C, C69S Trx1. To perform the enzyme assay, 20 nm TrxR1, 0.2 mm NADPH, the indicated amounts of ATG, the indicated amounts of ebselen, and 3 μm Trx1 were added into the reaction solution, and then reactions were initiated by the addition of 0.16 mm insulin. D, 4.5 μm pre-reduced TrxR1 was incubated with 10 μm ATG and/or 50 μm ebselen for 20 min. Then, 48 μm Trx1 was incubated with the mixture for 30 min. The reaction mixtures were then subjected to SDS-PAGE gel in the absence (lanes 1-4) or presence (lanes 5-8) of DTT. M, protein marker. E, the reaction mixtures were subjected to Western blot analysis using Trx1 antibody (lanes 1 and 2) and TrxR1 antibody (lanes 3 and 4).