(A) Schematic description of constructs used for the assay, with numbers denoting the amino acid residue of the protein. Both fluorescent proteins, CFP and YFP, are attached N-terminally to the IRE1 LD and BiP proteins, respectively. (B) Schematic representation of the in vitro FRET UPR induction assay, which measures a three-component protein interaction system. CFP-IRE1 LD will be excited at 430 nm with a bandwidth of 10 nm (430–10 nm), and upon excitement it will emit radiation at the longer wavelength of 480–10 nm. When YFP-BiP is added, IRE1 LD should interact with BiP via its ATPase domain, bringing YFP in close proximity to CFP, resulting in FRET between the fluorescent proteins and an emission at 530–10 nm. Dissociation of the complex—with loss of FRET signal—should occur upon addition of misfolded protein, which will bind to BiP substrate-binding domain (SBD) to cause conformational change. The ratio of 530-10 nm/480-10 nm will be used to measure FRET signal output. (C) Bar graph of the FRET ratio (530–10 nm/480–10 nm) upon excitation at 430–10 nm wavelength when CFP-IRE1 LD and YFP-BiP were mixed in equimolar amounts. This was compared to non-binding controls, YFP with CFP-IRE1 LD, and BiP with C-terminally tagged YFP with CFP-IRE1 LD. The FRET ratio was almost doubled upon interaction revealing a clear FRET signal. The negative controls measure a FRET ratio of ~0.34 due to CFP, which contributes a significant fluorescence emission intensity at 530 nm (also referred to as CFP leakage) when excited at 430 nm. This allows for greater spectral overlap with YFP making CFP and YFP excellent FRET pairs, but adds to the background noise. The data are shown as mean ± SD (n = 6). (D) FRET UPR induction assay measurements upon addition of misfolded protein CH1. In this graph, the FRET signal is represented as a percentage, with 0% observation equivalent to non-binding control and 100% being represented by CFP-IRE1 LD and YFP-BiP. The addition of CH1 caused IRE1 LD and BiP dissociation, resulting in the loss of FRET signal (mean ± SD; n = 6).