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. 2019 Feb 22;14(2):e0212711. doi: 10.1371/journal.pone.0212711

Fig 1. Synchronization of WNT3A protein using RUSH system.

Fig 1

(a) Schematic of the RUSH-Wnt system. Under physiological conditions, KDEL -streptavidin binds to SBP-XFP-Wnt to retain the protein in the ER. Palmitoleated Wnts bind to WLS in the ER and their release is induced by the addition of 100 μM biotin. The SBP-XFP-Wnt is transported to the Golgi and subsequently to the plasma membrane. (b) RUSH-modified WNT3A is signaling-competent. β-catenin-dependent luciferase expression was stimulated by transient expression of the indicated proteins in SuperTopFlash cells. 100 μM biotin was added where indicated 5 hours after transfection, and luciferase activity was measured ~16 hours later (**p<0.01). (c) Fluorescence photomicrographs of HeLa cells expressing RUSH-eGFP-WNT3A at various time points after biotin addition (from S1 Video). At time 00:00 (minutes:seconds) of biotin addition, WNT3A remained in the ER. At time 10:40, WNT3A starts to leave ER and can be seen in the Golgi. At time 17:20, WNT3A is seen entirely in Golgi. Starting around 28:20 and continuing at 35:00 WNT3A can be seen in vesicle exiting the Golgi and moving towards the periphery. By time 45:00, the Golgi begins to be depleted of WNT3A. (d) Time-dependent Golgi localization of RUSH-eGFP-WNT3A. The plot shows fluorescence intensity in the Golgi region (white box in Fig 1C) at different time points after biotin addition. Intensities were normalized to the maximal Golgi intensity with arbitrary units. (n = 7 independent cells ± SD).