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Fig. 4. Constitutive association of TrkA-Ros and SHP-1 in intact cells. (A) Association of TrkA-Ros and SHP-1 in HEK293 cells demonstrated by streak camera measurements. TrkA-Ros-ECFP was coexpressed with different EYFP-SHP1 variants as indicated. Fixed cells were excited at 430 nm and emitted fluorescence was recorded by a combination of a spectrograph and a streak camera. Typical examples of fluorescence emission spectra (upper panel) and fluorescence decay curves in the wavelength range from 465-495 nm (lower panel) that were calculated from the recorded data (streak images) are shown. Cumulated data are presented in Table 2. (B) Detection of constitutive interaction between TrkA-Ros-ECFP and EYFP-SHP-1 by fluorescence spectroscopy. TrkA-Ros-ECFP was coexpressed with the EYFP-SHP1-SH2 or EYFP-SHP1-R32K fusion proteins, as indicated, in COS7 cells. Cells were suspended and spectra were recorded in a fluorescence spectrophotometer with excitation at optimal wavelength for ECFP ({gamma}ex=424 nm). EYFP spectra ({gamma}ex=494 nm) were recorded in the same samples and revealed that the amount of EYFP constructs in both sets of cells was identical (not shown).





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