Fig. 5.

VSRs bind ligands after recycling. a Targeted VSR sensors (Nb_G-LDB) were shown to bind Aleu-RFP ligands in the Golgi²⁷ b GFP-SYN-labeled Nb_G-VSRs are locked to the anchor Man1-Nb_S in the cis-Golgi, positively identified by the marker Man1-BFP2. Ligand-binding of recycled full-length VSRs is assessed by colocalization with ligands (Aleu-RFP). Controls with cycling VSRs that lack c the anchor or d the SYN epitope at the labeling GFP for the VSR lockdown, result in VSR localization at the TGN/EE, which does not promote ligand binding. e GFP-SYN-labeled Nb_G-VSRs are locked after recycling in the cis-Golgi and colocalize with the Golgi marker Man1-BFP2 and bind the ligand Aleu-RFP, as shown by the overlapping signal peaks in the line intensity plot (see b). f, g Not locked VSRs (see c and d) do not localize to the Golgi and thus do not bind the ligand Aleu-RFP as judged by the separated peaks in the line intensity plots. Scale bars 10 µm, insets 5 µm, showing magnifications