Figure 3
From: Retarded PDI diffusion and a reductive shift in poise of the calcium depleted endoplasmic reticulum
Calcium concentration-dependent association of PDI1A and calreticulin in vitro . (A) Schema of the calcium-dependent fluorescence resonance energy transfer (FRET) between a green fluorescent protein tagged calreticulin (CRT-GFP) donor and a monomeric Cherry tagged PDI1A (PDI1A-mCherry) acceptor. (B) Photomicrograph of Coomassie-stained fusion proteins, before and after TEV protease cleavage of the GST tag, resolved on a 10% SDS-PAGE. (C) Trace of calcium concentration-dependent changes in FRET between the CRT-GFP donor and the PDI1A-mCherry acceptor (both at 12 μM). The half-maximal change in the FRET signal is observed at 190 μM free Ca2+. Shown is a typical trace of an experiment reproduced three times. (D) As in ‘C’ above, but substituting mCherry for PDI1A-mCherry as the FRET acceptor. PDI1A, protein disulfide isomerase 1A.