The DHFR tag does not perturb subcellular localization. (A) Subcellular localization of TfR-GFP (left) versus TfR-GFP-DHFR (right) in COS-7 cells (in the absence of zapalog) relative to the RE markers mCh-DHHC2 (top) and mCh-Rab11 (bottom). Insets show magnified images of the regions marked by the white box. Yellow arrowheads in insets denote colocalized puncta. Scale bar, 20 µm. Inset scale bar, 5 µm. (B) Quantification of the data shown in A. Colocalization between TfR-GFP or TfR-GFP-DHFR and the RE markers mCh-DHHC2 and mCh-Rab11 was assessed by calculating the proportion of either TfR-GFP (white bars) or TfR-GFP-DHFR (gray bars) that overlap with the respective RE marker; mean ± SEM (unpaired t test; P = 0.7346 [DHHC2], P = 0.4558 [Rab11]; n = 10 cells/condition). (C) TfR-GFP-DHFR (green) colocalizes with ER marker mCh-Sec61 (red) in COS-7 cells expressing FKBP-KDEL in the presence of zapalog. The white arrowhead denotes the nuclear envelope, which is contiguous with the ER and contains both red and green signals. Bottom: Enlarged images of mCh-Sec61 and TfR-GFP-DHFR at the cell periphery (denoted by dashed box in top panel) where ER morphology is distinct. A plot of pixel intensities along the red line (in blow-up panel of mCh-Sec61) is plotted to the right. Scale bar, 10 µm; magnified images are 6 × 6 µm. (D) Shown is a COS-7 cell expressing TfR-mCh-DHFR, the GA marker mEmerald-GalT, and FKBP-KDEL in the presence of zapalog before (left) and 15 min following 405-nm light exposure (right). The magenta arrowheads denote the GA. The insets show colocalization between mEmerald-GalT and TfR-mCh-DHFR. Note the display settings were adjusted for the TfR-mCh-DHFR 15 min after image to avoid apparent saturation of the signal as it concentrated in the GA. Pixel intensities along the red line shown in the upper left are plotted for mEmerald-GalT (green line) and TfR-mCh-DHFR before ER release (red dashed line) and 15 min after ER release (solid red line). Scale bar, 10 µm; magnified images are 5 × 5 µm.