Characteristics of BRET of CX3CL1 in HEK cells. A, HEK293
cells were transfected with 0.05 μg of CX3CL1-Luc and 0.5 μg of various
YFP constructs: empty pEYFP (left), CXCL16-YFP (middle),
CX3CL1-YFP (right). The values are the mean ± S.D. of 15
measurements. Insert, fluorescence microscopy imaging of HEK cells
transfected with CX3CL1-YFP (bar = 50 μm). B, HEK cells
were transfected with 1, 2, or 3 μg of CX3CLI-Luc or CX3CL1-YFP constructs.
The luminescence of fluorescence of 50,000, 100,000, and 200,000 cells was
then measured. In addition, the CX3CL1 content of each transfectants was
analyzed by SDS-PAGE and Western blot and evaluated with
CX3CL1-His6 as standard. C, BRET variation using HEK cells
transfected with a constant amount of donor (CX3CL1-Luc) and increasing amount
of acceptor (CX3CL1-YFP) (▪). CX3CL1-Luc and CX3CL1-YFP contents in each
sample were quantified with the calibration curve in B. Here the
CX3CL1-Luc was kept constant at 1.5 fmol/mg total protein. The same
measurements were taken with CXCL16-YFP as acceptor (□). The data were
fitted with GraphPad Prism 4 with one-site binding hyperbola. The resulting
BRETmax was 0.230 for CX3CL1-Luc/CX3CL1-YFP and 0.027 for
CX3CL1-Luc/CXCL16-YFP. D, BRET variation using HEK cells
co-transfected with CX3CL1-Luc and CX3CL1-YFP at a constant ratio ([YFP]/[Luc]
= 5). The linear regression is given and indicates that BRET is constant in
this range. E, BRET variation versus CX3CL1-YFP
concentration using HEK cells transfected with various amounts of CX3CL1-Luc
and CX3CL1-YFP. F, HEK293 cells were transfected with 0.1 μg of
CX3CL1-Luc and 0.2 μg of CX3CL1-YFP supplemented with 2 μg of empty
pcDNA3 (left) or CX3CL1-pcDNA3 (right). The values are the
mean ± S.E. of 15 measurements. The difference between the CX3CL1 BRET
ratio with or without native CX3CL1 was significant (***,
p < 0.0001). The CX3CL1 content of each sample was analyzed by
Western blot to check that the untagged CX3CL1 expression did not change
expression of CX3CL1-Luc and CX3CL1-YFP (data not shown).