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. 2015 Jun 8;209(5):687–704. doi: 10.1083/jcb.201409127

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© 2015 Ungricht et al.

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Figure 2. — The INM targeting assay. (A) Schematic representation of INM targeting reporters used in this study. (B) Release of INM reporters from the ER to the NE upon TEV cleavage in vivo. Tetracycline-inducible HeLa cell lines expressing 2×RFPtevSUN2(fl)-GFP, 2×RFPtevLBR(1–245)-GFP, or 2×RFPtevLAP2β-GFP were transiently transfected with CFP-TEV protease. Localization of reporters was analyzed after 24 h. (C) Reconstitution of INM targeting in semipermeabilized cells (perm) was initiated by TEV protease cleavage for 10 min at RT (perm, TEV) and started by supplementation with HeLa lysate and an energy regenerating system. At the indicated time points, cells were fixed and analyzed by confocal microscopy. Bars, 10 µm.