Fig. 2: Enzymatic removal of fluorescent signal (EnzyPlex).

a Schematic of EnzyPlex cleaving process. Pre-formed complexes of 1.Ab and fluorescent Enzy-2.Nbs can be cleaved with bdSENP1 protease. b SDS-PAGE of different Enzy-2.Nbs before (-) and after (+) the addition of 1 μM bdSENP1. c Confocal images from U2OS-Nup96-GFP cells labeled against vimentin (magenta) using complexes of 1.Abs and Enzy-2.Nb carrying the Atto643. The sample was treated with 1 μM of bdSENP1 for 15 min, and vimentin’s fluorescent signal was recorded at 0 (before cleavage), 2.5, 5, 10, and 15 min upon bdSENP1 application. d The fluorescent intensities at each time-point were normalized to the intensity values before the application of bdSENP1. e Confocal 6-Plex image from U2OS-Nup96-GFP cells undergoing 3 iterative cycles of EnzyPlex and 2-channel confocal imaging per cycle. Bar graph displays the mean ± SD of the signal removal efficiency for each target (n = 3 cells). The NbALFA-Atto643 revealed tubulin and vimentin, while NbALFA-AZDye568 fluorophore was used for clathrin, peroxisomes, and nuclear speckles. GFP signal was acquired from Nup96-GFP (NPC). Plot profiles depict the fluorescence intensity across the denoted line for each target before and after signal removal. Staining details are in Methods and Supplementary Table 4. f 5-EnzyPlex dSTORM image from U2OS cells upon 5 iterative cycles. The Janelia Fluor JF635b was employed for single molecular localization imaging of alpha-tubulin (1), clathrin (2), vimentin (3), peroxisomes (4) and Nup96-EGFP on nuclear pores (5) with average localization precision (nearest neighbors distance estimator-NeNa) of 10.2, 10.6, 14.5, 13.3 and 16 nm, respectively. Details on stainings are in Supplementary Table 5.