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. 2019 Oct 10;10(10):771. doi: 10.1038/s41419-019-2011-5

Fig. 3. The reverse RUSH system as a tool for tethering reporters to subcellular structures.

Fig. 3

a Scheme depicting the principle of the reverse RUSH system. In presence of biotin, streptavidin is bound by biotin preventing interaction with SBP. Upon addition of excess avidin, biotin is titrated out and SBP can bind to streptavidin. Using different fusion constructs of SBP and streptavidin, a protein of interest (here GFP) can be directed to different subcellular structures. b Immunofluorescence staining of cell lines stably expressing ER hook and different GFP baits. Streptavidin staining, GFP signal, CALR staining and merge in the presence of biotin (40 µM), and after shifting to avidin (60 µM) for 12 h are shown. Scale bar equals 10 µm. c As in (b), but for cell lines expressing Golgi hook, and stained for B4GALT1 instead of CALR. d Quantification of relative co-occupancy of CALR immunofluorescence signal with SBP–GFP–LC3 (left) or p62–SBP–GFP (right) from images shown in (b) at indicated time points after shifting to avidin, and at 12 h washout after 24 h incubation in presence of avidin (wash). e As in (d), but for images shown in (c) and staining for B4GALT1. Bars indicate means ± standard deviation of at least three replicates (*p < 0.05, **p < 0.01, and ***p < 0.001, two-tailed Student’s t test, compared to cells before avidin addition; #p < 0.05, two-tailed Student’s t test, compared to cells after avidin washout)