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Fig. 5. Ligand-stimulated complex formation between SHP-1 and TrkA-Ros in COS7 cells and effect of binding site mutations. COS7 cells expressing TrkA-Ros-ECFP, or TrkA-Ros-ECFP mutants together with EYFP-SHP1-SH2 as indicated, were starved in serum-free medium for 6 hours and were either left untreated or were stimulated with 100 ng/ml NGF for 20 minutes. Thereafter, the cells were fixed and subjected to fluorescence lifetime imaging. (A-D) Left panels: fluorescence lifetime images. Amplitude weighted mean lifetimes (
m) were color coded as indicated in the right panels. Right panels: distribution of mean lifetimes over the entire cell. The mean fluorescence lifetime 
was calculated by averaging over all pixels of the cell taking into account their relative intensity. (E) Bar graph of the mean of the fluorescence lifetimes () determined in 5-11 cells. White bars represent measurements in the absence, and grey bars in the presence of NGF. Error bars represent the standard error of the mean. Significant (Student's t-test, P<0.05) differences between the data sets are marked by brackets. Green brackets mark a significant decrease of mean lifetimes that can be attributed to NGF. Dark and light blue brackets indicate data sets that reflect significant changes in constitutive association.
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