Fig. 7. Loss of perinuclear ERES in TBC1D20-expressing cells. (A) HeLa cells transfected with GFP-tagged wild-type TBC1D20 or a catalytically inactive R105A point mutant (green) were fixed after 22 hours and then stained with antibodies to the COPII subunit Sec31 (red). DNA was stained with DAPI (blue). The clustering of COPII structures in the perinuclear region, or TBC1D20-induced scattering was counted and the results are show in the bar graph below (100 cells per experiments, n=2). Bar, 10 µm, in all panels. (B) FRAP was performed on cells expressing Myc-TBC1D20 and Venus-Sec16 using a Leica TCS SP3 AOBS scanning confocal microscope with a pinhole size of 2 Airy units, eightfold line averaging, at 1 frame per second, and region-of-interest bleaching with 10 iterations of the 514 nm laser at 100% AOTF power. Cells used for photobleaching were confirmed to express Myc-TBC1D20 by subsequent immunofluorescence; 95% of all cell transfected with Venus-Sec16 were found to be transfected with Myc-TBC1D20. Images taken from the time points indicated were quantified using ImageJ. Plot of the average intensity of individual structures over time, error bars indicate the standard deviation (five cells per experiment, n=3).