(A) Visualization of transferrin-Alexa488 (green) and EGF-Alexa647 (magenta), as well as the transfected protein, visualized with the ALFA nanobody (NbALFA) conjugated to AZdye568 (white). The 3 rows show the 10-minute pulse with the ligands (endocytosis), followed by the 10- and 20-minute chase (wash-off). To enable optimal visualization, the images are scaled differently, with the image scaling indicated in all panels. Scale bar: 20 μm. (B) The nanobarcoding scheme and the expected localization of the protein. (C) The NbALFA fluorescence intensity is plotted against the transferrin (green) and EGF (magenta) intensity, for all signals measured in 2 independent experiments, for all conditions. All intensities were normalized to the medians of the distributions and were then grouped in 20 bins of ALFA intensity, each containing similar numbers of values. The mean and SEM of each bin in the respective channels are plotted. The data underlying this Figure can be found in the S1 Data file, Sheet “SFig 15C_VAMP4,” available from http://dx.doi.org/10.17169/refubium-40101. (D) The Pearson’s correlation coefficients for the distributions from panel C are shown, with the p-values corrected for multiple testing using a Bonferroni correction. (TIF)</p

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