NE protein recruitment is not delayed by Rtn4 overexpression. A real-time microscopic assay was established to measure the recruitment of GFP fusion proteins to the forming NE. (A) U2OS cells were transfected with H2B-tdTomato and Sun1-GFP with or without the overexpression of V5-Rtn4 and imaged every 30 s through mitosis. A thin region surrounding the chromatin (NE region) was selected to measure the intensity of Sun1-GFP at the forming NE. (B) Fluorescence intensity of POM121-3GFP was measured at the NE region in control cells (red) or cells expressing V5-Rtn4 (green) starting at the onset of chromosome segregation, then normalized, averaged, and plotted with standard error bars. (C) Cells expressing Sun1-GFP were analyzed as in B. (D) U2OS cells were grouped for high (red) and low (green) expression of GFP-Sec61, and fluorescence intensity was measured at the forming NE as in A. (E) Cells expressing GFP-Rtn3 were analyzed as in D. (F) Cells expressing DP1-GFP were analyzed as in D. Interphase localization of each GFP fusion protein is shown in the insets in B–F. Bars: (A) 10 μm; (B) 5 μm.