FRET on HA and Myc tagged ectodomain constructs in HEK 293 cells.

<p>HA-GPI-TSHR-ecd and myc-GPI-TSHR-ecd were co-transfected into HEK 293 cells and stained with anti-HA FITC and anti-Myc Cy3 labeled antibody and imaged for energy transfer between FITC (donor) and Cy3 (acceptor) fluorophores. As described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009449#s4" target="_blank">Materials and Methods</a> single transfected cells labeled with anti-HA-FITC and anti-Myc-Cy3 alone were the endogenous control for lack of FRET and for correction of donor and acceptor spectral bleed through. <b>Panel A</b> shows the staining of GPI-TSHR-ecd by anti HA FITC. <b>Panel B</b> shows the same cell stained with anti Myc Cy3. <b>Panel C</b> is the pseudo colored FRET energy level image obtained from the analysis of the double transfected cells by using the pFRET software. The calibration bar indicates the FRET energy t level in the cell. Energy transfer was in the range of 8–10% on the surface of the cells compared to controls (TSHRGFP +TSHRmyc labeled with Cy3) which showed energy transfer of 24%.