RAB6 is not involved in sorting of cargos at the exit of Golgi complex. (A) HeLa cells coexpressing SBP-EGFP-ColX and TNFα-SPB-mCherry, SBP-EGFP-ColX and SBP-mCherry-CD59, or TNFα-SBP-EGFP and SBP-mCherry-CD59 were incubated for 35 min with biotin to allow cargo release from the ER and then imaged using time-lapse video microscopy. Representative images taken from videos are shown (higher magnification on the right). Orange arrowheads point at colocalized vesicles; white arrowheads point at non-colocalized vesicles. Right: Quantification for each pair of cargos of the percentage of vesicles positive both cargos (yellow), EGFP (green), or mCherry (red; mean ± SEM, n = 197–501 vesicles from 11–20 cells). (B) Staining of endogenous RAB6:GTP (blue) in cells coexpressing SBP-EGFP-ColX (green) and TNFα-SBP-mCherry (red) and incubated for 30 min with biotin to visualize post-Golgi carriers. Higher magnifications are shown on the right. Quantification of the percentage of vesicles containing both SBP-EGFP-ColX and TNFα-SPB-mCherry (yellow), only SBP-EGFP-ColX (green), or only TNFα-SPB-mCherry (red). Dashed bars indicate the percentage of vesicles positive for RAB6 (mean ± SEM, n = 24–128 vesicles). Scale bars, 5 µm.