There is no spillover of signals between the detectors that measure DiD and EYFP.
Transfection efficiencies were visualized by means of the EYFP tag fused to the LDLR, and any differences were allowed for in the calculations.
To determine whether the cellular localization of LDLR was affected by introduction of an EYFP tag, we evaluated CHO cells expressing wild-type LDLR, with or without EYFP tag, by confocal microscopy (Fig.
After having verified that the EYFP tag did not affect intracellular traffic or receptor function, we used the fusion protein to develop methods for characterizing LDLR sequence variations in transfected cells.
1] Nonstandard abbreviations: FH, familial hypercholesterolemia; LDLR, LDL receptor; ER, endoplasmic reticulum; EYFP, enhanced yellow fluorescent protein; T-Rex, tetracycline-regulated expression; FBS, fetal bovine serum; DiI, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; PBS, phosphate-buffered saline; BSA, bovine serum albumin; mAb, monoclonal antibody; EEA1, early endosome antigen 1; FITC, fluorescein isothiocyanate; and DiD, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate.
The fluorescence of EYFP was detected with an excitation filter of 485 nm and an emission filter of 545 nm, and that of ECFP was detected with filters of 420 and 486 nm (Perkin Elmer), respectively.
In the present reporter assay, EYFP and ECFP were selected as a reporter protein and an internal control for normalization of transfection, respectively.
However, the apparent rate of the response was enhanced by interference of the expression of ECFP by the expression of EYFP, because the same promoter was employed for the reporter plasmid and the internal control plasmid.
Finally, we established the following plasmid set as the reporter assay system: a reporter plasmid containing the EYFP gene, whose expression was regulated by the HRE and CMV201 promoter; an internal control plasmid containing the ECFP gene expressed by the SV40 promoter; and the expression plasmid of the nuclear receptor containing each nuclear receptor gene expressed by the CMV promoter.