Figure 5.

Stable association of class II Arfs with the ERGIC in the presence of BFA does not require GBF1. (A) Images correspond to the 10-min time points from the Arf4 and Arf5 movies shown in Figure 3A. Bottom panels show single and merged images acquired in the boxed area from the mCherry and GFP channels. Representative of at least three experiments in NRK-GFP-GBF1 cells. Bar, 10 μm for the full images and 2 μm for insets. (B) NRK-GFP-GBF1 cells transfected with either Arf4-mCherry (top row) or Arf5-mCherry (bottom row) were incubated on ice for 2 min followed by treatment with 20 μg/ml NOZ for 15 min on ice. Several minutes (5–10 min) after transfer to a heated stage, cells were treated with BFA and imaged every 5 s for 10 min. Images correspond to frames captured at the indicated times. Bottom panels show single and merged images acquired in the boxed area from the mCherry and GFP channels. Images representative of at least four experiments in NRK-GFP-GBF1 cells. Bar, 10 μm. (C) NRK cells were cotransfected with plasmids encoding p58-GFP, and either Arf4-mCherry (top) or Arf5-mCherry (bottom) and then treated and imaged as described in B. Arf4/5-mCherry–positive structures after treatment with NOZ and BFA contain p58-GFP and correspond to the ERGIC. Images representative of at least two experiments in NRK cells. Bar, 10 μm.