Extended Data Fig. 1. Nanobody Specificity and Fluorescence Background.
a, Specificity of the LaG-9 anti-GFP nanobody. Alexa568-labeled nanobody (100 nM) was added to permeabilized cells, incubated for 3 min and washed twice with IB + PVP. Cells were imaged at the nuclear equator. The top row shows the strong GFP fluorescence (λex = 488 nm) obtained from the mEGFP-tagged NPCs of U-2 OS NUP96-mEGFP cells. The Alexa568 fluorescence (λex = 561 nm) from the tagged nanobody matches the GFP fluorescence. In the bottom row, the wild type U-2 OS cells exhibited no fluorescence in the GFP channel and no nanobody binding. The range of the fluorescence images in the bottom row is 10 times smaller than in the top row, emphasizing the very low signal. These data indicate the high specificity of the nanobody for the GFP domain. Similar results were observed for N = 20 cells over 4 independent experiments. b, Fluorescence background and photobleaching of mEGFP. For all images, permeabilized U-2 OS NUP96-mEGFP cells labeled with HMSiR-labeled nanobodies were excited with λex = 532 nm and emission was collected by a dual bandpass (545-623 nm, 656-763 nm). The mEGFP fluorescence (left) was photobleached after ~2 s of illumination, allowing diffusing M9-βGal(Atto542) cargo complexes to be readily visualized (top right). The HMSiR dye was not detectable (bottom right). Similar results were observed for N = 20 cells over 4 independent experiments.