Fig. 1: Engineering human cell lines to study native COPII dynamics.
a Representative immunoblots (n = 3) of extracts generated from control and clonal CRISPR/Cas9 edited cells using antibodies directed against TFG (top) and GAPDH (bottom). b Representative confocal images of cell lines natively expressing HaloTag fusion proteins after labeling with JFX650-HaloTag ligand are shown. Insets show 3x zoomed regions (boxed). Bar, 10 μm. c Representative super resolution images of fixed cells expressing HaloTag fusion proteins labeled with the JFX650-HaloTag ligand (magenta) and co-stained using antibodies directed against Sec24a (green). Color-coded linescans highlighting their relative localizations are shown (right). Bar, 500 nm. d Confocal imaging of edited cell lines (HaloTag-Sec16a, green; HaloTag-Sec23a, light blue; HaloTag-Sec31a, purple; HaloTag-TFG, salmon) co-expressing either ss-DsRed (top) or ManII-SBP-GFP (bottom) was used to monitor their synchronous release from the ER. Based on fluorescence intensity, the percentage of each cargo present within the perinuclear region relative to its maximal accumulation there was quantified over time. Error bars represent mean +/- SEM (n = 20 cells each; 3 biological replicates each). ***p = 0.0003, **p = 0.0082, and *p = 0.0260, as calculated using a two-way ANOVA and Dunnett’s multiple comparison test. e Confocal microscopy was used to monitor the fluorescence recovery of HaloTag-Sec16a labeled with JFX650-HaloTag ligand after partial photobleaching (n = 20; 3 biological replicates). Error, as displayed by green-colored bands around the smoothed tread line, represents mean +/- SEM. Based on the recovery curve, the mobile fraction (Mf) and half-time of recovery (t1/2) were determined. Source data are provided as a Source Data file.
