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Fig. 4. Analysis of gp130 homodimerization and gp130/LIFR heterodimerization by FRET. (A) Hek293T cells were transfected with expression vectors encoding gp130/id-CFP and gp130/id-YFP. 48 hours after transfection, cells were stimulated with IL-6 (20 ng/ml) and sIL-6R{alpha} (1 µg/ml) (red squares) or left unstimulated (blue diamonds). 15 minutes after stimulation, cells were fixed and analysed by quantitative FRET at the confocal microscope. Individual FRET efficiencies at the plasma membrane of cells expressing gp130/id-CFP and gp130/id-YFP are given as a function of the excess of YFP (acceptor) fluorescence (left) or as a function of the average fluorescence intensity of CFP and YFP (right). Individual cells were selected for each FRET measurement. (B) Mean values of the individual FRET efficiencies at the plasma membrane of unstimulated (light grey) or stimulated (black) cells expressing gp130/id-CFP and gp130/id-YFP in comparison to FRET negative and positive control (±s.d.). Significant differences (***P<0.001) in FRET efficiency were observed between experimental groups and the two control groups as indicated. Dashed bars represent the calculated FRET efficiencies for exclusive heterodimerization between CFP and YFP. (C) Hek293T cells were transfected with expression vectors encoding human LIFR/id-CFP and gp130/id-YFP. 48 hours after transfection, cells were stimulated with LIF (20 ng/ml) (red squares) or left unstimulated (blue diamonds). 15 minutes after stimulation, cells were fixed and analysed by quantitative FRET at the confocal microscope. Individual FRET efficiencies at the plasma membrane of cells expressing LIFR/id-CFP and gp130/id-YFP are given as a function of the excess of YFP (acceptor) fluorescence (left diagram) or as a function of the average fluorescence intensities of CFP and YFP (right diagram). Individual cells were selected for each FRET measurement. (D) Mean values of the individual FRET efficiencies at the plasma membrane of unstimulated (light grey) or stimulated (black) cells expressing LIFR/id-CFP and gp130/id-YFP in comparison to FRET negative and positive control (±s.d.). Significant differences in FRET efficiency (***P<0.001) were observed between groups as indicated.





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