We finally tested alternative redox-active dithiol proteins. We first profiled dose-response to Grx1 and Grx2, the other major vicinal dithiol protein redox effectors, applied as part of the full GR-GSH-Grx cascades (Fig. , Fig. ). Both RX1 and G1 fully resisted up to 10 µM Grx1/Grx2 over 3 hours (including 20 nM GR, 200 µM NADPH, and 100 µM GSH for Grx reduction). This is a significant result. Although the Grxs are vicinal dithiol redox enzymes similar to the Trxs, they are more RSSG-selective. Therefore the cyclic probes should resist Grx processing inasmuch as they resist monothiol exchange (note too that although GR is NADPH-driven, it has very high specificity for GSSG so is not expected to directly reduce the probes). We also examined the TrxR-reduced redox-active vicinal dithiol TRP14, finding that the probes resisted it up to 3-10 µM (Fig. , Fig. , Fig. ). These results show that the probes are not sensitive to all vicinal dithiol proteins per se, but have a specific sensitivity to the highly reducing Trxs at least in cell-free conditions. While it would have been best for our TrxR-reporter goal if Trxs were incapable of probe reduction, sensitivity can be expected in a cell-free assay where excess NADPH and an absence of other oxidants and binding partners offers no alternative pathway except probe reduction. In cells however, Trx as an effector protein has many binding partners that recognise and oxidise its thioredoxin fold, while NADPH-powered TrxR binds fewer substrates (mainly Trx, also TRP14, and potentially Grx2). This gave hope that if Trx in cells would be substantially engaged with preferred protein substrates, then RX1's extraordinary kinetics of turnover by TrxR1, combined with its total stability to GSH, might give it functional TrxR1-selectivity in cells. In line with this understanding, previous research on Trx has identified cyclic dichalcogenide probe chemotypes that were highly sensitive to purified Trx (but not to TrxR) in cell-free settings, yet were unexpectedly inactive in cellular settings despite the high cellular concentrations of this universal reductant. 17