Association of PDI1A and calreticulin under physiological conditions of calcium depletion. (A) Donor (cyan fluorescent protein) fluorescent intensity and donor life time images (FLT) of D1ER Cameleon in untreated and cholecystokinin (CCK)-treated (10 minutes) pancreatic acinar AR42j cells. A histogram of the distribution of lifetimes is provided (right) noting the mean ± SD (vertical white lines) lifetime of the cells in the sample. Further to the right is a pseudo-colored image of calcium concentration derived from Cameleon’s life time and notation of the mean of the concentration in each sample. Note the decline in mean [Ca2+]ER from 2.6 mM in the untreated to 80 μM in the sample exposed to the highest concentration of CCK. The size bar is 20 μm. (B) Donor (GFP) fluorescent lifetime of GFP transfected alongside mCherry, setting the lower limits of the FRET for this pair in AR42j cells. (C) Donor (GFP) fluorescent lifetime of a fusion protein of GFP and mCherry, setting an upper limit of FRET for this pair in AR42j cells. (D) Donor (GFP) fluorescent lifetime of CRT-GFP fusion protein transfected alongside and ER mCherry, as a FRET acceptor. Where indicated the cells were treated with CCK (10 μM for 15 minutes). (E) Donor (GFP) fluorescent lifetime of CRT-GFP fusion protein transfected alongside PDI1A-mCherry, as a FRET acceptor. Samples were treated with CCK (10 μM) for the indicated time. (F) Bar diagram of fraction of GFP probe bound to mCherry probe derived from the FRET measurements in B-E (mean ± SEM, n >100, P <0.001). The size bar is 20 μm. CRT, calreticulin; FRET, fluorescence resonance energy transfer; PDI1A, protein disulfide isomerase 1A; SD, standard deviation; SEM, standard error of the mean.