Fig. 4
Thapsigargin and cyclosporine A increase glutathione levels in the ER lumen. a Formula for the calculation of [GStot]ER from [GSH]2:[GSSG] and [GSH]:[GSSG] in the ER. b [GSH]2:[GSSG] was quantified in the ER of Grx1-roGFP1-iEER-expressing HEK293 cells that were left untreated (−) or treated with TG or CsA for 15 min by measuring the ratiometric emission intensity values of Grx1-roGFP1-iEER at steady state, fully oxidized, and fully reduced conditions. c sCGrx1pER-transfected HEK293 cells were left untreated (−) or treated with TG or CsA for 15 min. Glutationylation state ([−SH]:[−SSG]) of sCGrx1p was determined by immunoprecipitation and TMMPEG modification of the radiolabelled protein. [−SH]:[−SSG] was quantified by SDS-PAGE, phosphor imaging, and densitometric analysis. Samples obtained from cells that were treated with 10 mM DTT or 5 mM diamide (dia) served as mobility markers for −SH and −SSG, respectively. The vertical dashed line indicates where an intervening lane has been removed. Note that [GSH]:[GSSG] is directly proportional to [−SH]:[−SSG]. One of three representative experiments is shown