FIG. 3.
Specificity of localization of VE-DSR and GFP-MyoX in double-fluorescently transfected HUVECs. To control the specificity of GFP-MyoX localization, HUVECs coexpressing either GFP-MyoX and VE-DSR (panels at left) or GFP-MyoX and DSR (middle panels) were analyzed in green and red fluorescence channels. Using ImageJ, the fluorescent intensities (I) of GFP-MyoX spots (delimited by ellipses) were measured and compared to the fluorescent intensities of the superimposed areas in the other channel. While the mean value for the IGFP-MyoX/IVE-DSR ratio ranges around 1 (20 measurements from 5 images), that of the IGFP-MyoX/IDSR ratio varied between 3 × 105 and infinite (20 measurements from 5 images). Similarly, to control the specificity of VE-DSR localization, HUVECs coexpressing GFP and VE-DSR were analyzed in parallel for fluorescent localization (panels at right). While the mean value for the IVE-DSR/IGFP-MyoX ratio ranges around 1, that of the IVE-DSR/IGFP ratio varied between 1 × 105 and infinite (20 measurements from 5 images). From ratio-metric quantification, it can be deduced that the localization of VE-DSR or GFP-MyoX is not disturbed by GFP- and DSR-associated fluorescence, respectively.