Calcium insensitivity of the core disulfide oxidative machinery of the ER. (A) Trace of time-dependent changes in fluorescent lifetime (in picoseconds) of ER-localized roGFPiE preceding and following a dithiothreitol (DTT, 5 mM) pulse and washout in untreated (green) and thapsigargin (TG)-treated (orange) HEK 293 T cells. Each data point represents the mean ± SD of fluorescence lifetime measured in ≥8 cells. Note the shortening of the lifetime and slowness of its recovery following DTT reduction in the thapsigargin-treated group. A comparison of the mean half-time to recovery in the untreated and treated cells is provided in the bar diagram to the right (n >10, P <0.05%). (B) Plot of the initial velocity of roGFPiE oxidation in vitro (measured by the ratio of excitation intensity at 470 nm and 395 nm) in the presence of varying concentration of PDI1A, in a glutathione redox buffer (5:1 GSH/GSSG, 4 mM total, no added Ca2+). Kinetic parameters were extracted by fitting the data to a non-linear regression. (C) As in ‘B’, with PDI1A held at K0.5max (16 μM) and varying concentration of Ca2+. (D) As above, but in the presence of a saturating concentration of PDI1A (40 μM) and varying concentration of ERO1 (no added Ca2+). (E) As above, but in the presence of a saturating concentration of PDI1A (40 μM), ERO1 at K0.5max (0.5 μM) and varying concentration of Ca2+. ER, endoplasmic reticulum; PDI1A, protein disulfide isomerase 1A; SD, standard deviation.