29 research outputs found

    A functional assay to test nanobarcoded proteins.

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    Cells expressing different nanobarcoded proteins were pulsed with transferrin conjugated to Alexa488 and with EGF conjugated to Alexa647, for 10 minutes, allowing the cells to endocytose these ligands. Afterwards, they were immediately fixed or were chased (washed off) in a minimal buffer at 37°C, for 10 or 20 minutes. Finally, all cells were fixed and immunolabeled for the ALFA tag, to identify the nanobarcoded proteins. (A, B) The behavior of transferrin and EGF, respectively. Transferrin recycles, as expected, being released during the chase period (Kruskal–Wallis test followed by Tukey post hoc test, p N = 17–18 independent experiments. (C, D) Same data as above, but indicating the nature of the nanobarcoded protein in each of the independent experiments. The data underlying this Figure can be found in the following Sheets of the “S1 Data file: “Tf_SFig 6A,” “EGF_SFig 6B,” “Tf_SFig 6C,” and “EGF_SFig 6D.” The S1 Data file is available from http://dx.doi.org/10.17169/refubium-40101. (TIF)</p

    Endogenous SNAP25 and SNAP25(1100) have a similar cellular distribution within SNAP25(1100) transfected PC12 cells.

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    (A) Visualization of both endogenous SNAP25 and SNAP25(1100) using SNAP25 specific primary and secondary antibodies, plus NbALFA. (B, C) Negative control experiments, leaving out either primary antibodies (B) or NbALFA (C). (D) Imaging control, using a mixture of the same secondary antibody with 2 distinct fluorophores (targeting both endogenous SNAP25 and SNAP25(1100)), to provide a visual indication of the maximum expected colocalization. Bottom part of the figure: legend for used symbols and schemes. Scale bars: 2.5 μm. For quantification, see S17 Fig. (TIF)</p

    Transferrin and EGF imaging assays, tested for nanobarcoded syntaxin 13.

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    (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 14C_STX13,” 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

    Visualization of 15 nanobarcode epitopes using 4 spectrally distinct nanobodies.

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    (A-D) Nanobody-based identification of the 4 genetically encoded nanobarcode epitopes mCherry(Y71L), GFP(Y66L), syn87, and syn2 and the ALFA-tag epitope by their corresponding nanobodies NbRFP, NbEGFP, NbSyn87, NbSyn2, and NbALFA. Scaling was optimized for each protein. (D) VAMP4(1111) example (all epitopes present) and negative control condition: mock transfection (no DNA, no epitopes present) using same intensity scale. (E) As in (D), now with upscale intensities. Scale bar: 50 μm. (TIFF)</p

    Transferrin and EGF imaging assays, tested for nanobarcoded Vti1a.

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    (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 bars: 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 7C_Vti1a,” 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

    Visualization of nanobarcoded proteins that act as markers for specific organelles.

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    The proteins indicated in the left-most column are markers for specific compartments, indicated in the next column. The colocalization of these proteins and specific compartment markers is then indicated in the fluorescence images. Scale bars: 20 μm. For quantification, see S17 Fig, below. (TIF)</p

    Transferrin and EGF imaging assays, tested for nanobarcoded endobrevin.

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    (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 11C_Endo,” 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. (PNG)</p

    Validation of transfection and expression of protein constructs within HEK293 cells.

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    To enable an analysis of Nrxn and Nlgn pairing, we coexpressed different Nrxn and Nlgn constructs with specific nanobarcode proteins. This enables us to provide the different Nrxn- and Nlgn-containing cells with a recognizable identity, without having to modify the additional proteins themselves by nanobarcode tagging. However, this implies that we need to verify whether the majority of Nrxn- or Nlgn-expressing cells also express the respective nanobarcode proteins. (A, B) Nanobody staining with anti-ALFA-Atto488 reveals cells expressing protein constructs with nanobarcodes. Antibody staining with mouse-anti-HA and Cy3-anti-mouse reveals cells expressing NRXN or NL constructs with HA-tags. An overlay of both signals (anti-ALFA in green and anti-HA in magenta) indicates double-transfected cells, which make up the majority of all cells. N = 2 independent experiments for each protein combination. Scale bars: 50 μm. (TIF)</p
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