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. Author manuscript; available in PMC: 2020 Aug 17.
Published in final edited form as: Nat Chem Biol. 2020 Feb 17;16(3):327–336. doi: 10.1038/s41589-020-0474-4

Figure 1. Retro-2 binds directly to Sec16A.

Figure 1

(a) Scheme of biorthogonal click chemistry adapted to Retro-2.1. The clickable Retro-2.1 (compound 2), based on Retro-2.1 (compound 1, Ref.34, 50) is coupled via the DIBO moiety to biotin (compound 3), or a fluorophore (compound 4). Blue/dashed boxes indicate reactive centers. (b) Anti-Sec16A western blot of a representative pull-down with the clickable Retro-2.1-probe (compound 5). Load corresponds to the western blot of the input fraction. See Supplementary Fig. 7a for full gels. (c) Quantification of four (control and Retro-2.1) and two (competition, Comp.) independent experiments of clickable Retro-2.1 pull-down shown in (b). Bead control and competition conditions normalized to Retro-2.1 pull-down. Graph shows mean +/- SD, each point represents an individual experiment. Unpaired two-tailed t-test comparing Retro-2.1 to control, *** P < 0.0001. (d) Characterization of the Sec16A1266-1678/Sec13-Retro-2.1 interaction using bio-layer interferometry. Association and dissociation steps after subtraction of control curves (loading step without biotinylated Retro-2.1 probe) and analysis of binding kinetics of Sec16A1266-1678/Sec13 to biotinylated Retro-2.1 (compound 5). Graph is representative of 2 independent experiments. Data were acquired repeatedly from the same sample. (e) Confocal images of clickable Retro-2.1 reacted with an Alexa488 probe (compound 6) on mock siRNA transfected (control) or Sec16A-depleted (siSec16A) cells. DNA was stained with DAPI. Scale bars = 10 μm. Image representative of 2 independent experiments. (f) Quantification of fluorescence intensity of clicked Retro-2.1 (compound 6) in the indicated conditions. Quantification of 97 (negative control), 114 (Retro-2.1) and 77 (siSec16A) cells per condition from 2 independent experiments. Each point represents one cell. Negative control is fluorophore in the absence of clickable Retro-2.1 (“No Retro-2.1”). Graph shows mean +/- SD. Unpaired two-tailed t-test performed to compare Retro-2.1 treated and Retro-2.1+siSec16A conditions, *** P < 0.0001. See Supplementary Notes for numbered compound synthetic procedures.