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. 2008 Oct 31;283(44):30225–30234. doi: 10.1074/jbc.M802638200

FIGURE 2.

FIGURE 2.

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).