Mobility of ERdj5, a reducing member of the PDI family, is unaffected by ER calcium depletion. (A) Photomicrographs of immunostaining of endogenous BiP (an ER marker green), ERdj5-mCherry fluorescence (red) and an overlay of the two (yellow) in COS7 cells transfected with a plasmid encoding an ER-localized ERdj5-mCherry (Pearson’s coefficient of BiP/PDI1A-mCherry co-localization, overlap coefficient r = 0.82, Pearson’s coefficient r = 0.73). The purple Hoechst stains the nucleus. The size bar is 20 μm. (B) Trace of time-dependent changes in the normalized intensity of ERdj5-mCherry after photobleaching a small patch of transfected COS7 cell volume. The green trace is of an untreated sample and the orange of cells exposed to thapsigargin 10 minutes before data acquisition. The mean ± SD of the half-time to recovery of the two samples is provided. (C) Donor (GFP) fluorescent lifetime of CRT-GFP fusion protein transfected alongside ERdj5-mCherry, as a FRET acceptor. Where indicated the sample was treated with thapsigargin. (D) Donor (GFP) fluorescent lifetime of CRT-GFP fusion protein transfected alongside ER-localized mCherry, as a control FRET acceptor or fERdj5-mCherry. (E) Donor (GFP) fluorescent lifetime of a GFP-mCherry fusion protein setting the upper limit for FRET. The mean ± SD (vertical white lines) lifetime noted on the histograms. CRT, calreticulin; ER, endoplasmic reticulum; FRET, fluorescence resonance energy transfer; PDI1A, protein disulfide isomerase 1A; SD, standard deviation.