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. 2005 Mar 28;168(7):1039–1051. doi: 10.1083/jcb.200409138

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Figure 6. — Membrin “protects” Arf-1 from BFA-induced redistribution. (A–C) COS-7 cells overexpressing myc-membrin and Arf-1-GFP or Arf-1-6-1-GFP (A), myc-membrin and Arf-1-HA (B), or Arf-1-GFP and myc-rbet1 or HA-syntaxin5 (C) were untreated (Unt) or incubated in the presence of 5 μg/ml BFA for indicated times and fixed. Cells were immunolabeled with antibodies against myc (A), HA and β-COP (B), or myc or HA (C) followed by Alexa 594 anti–mouse (A and C), or Alexa 488 anti–mouse and Alexa 594 anti–rabbit (B) antibodies. Bars, 10 μm. (D and E) HeLa cells overexpressing Arf-1-GFP alone (D, control) or Arf-1-6-1-GFP alone (E, control), or with myc-membrin or myc-rbet1, or HA-syntaxin5 were untreated or incubated in the presence of 5 μg/ml BFA for 2 min and fixed and processed for immunolabeling with antibodies against myc or HA followed by Alexa 594 anti–mouse and anti–rabbit antibodies, respectively. To quantify Golgi and non-Golgi pools of Arf-1-GFP, one region of interest was drawn around Golgi and the other region of interest was drawn around the rest of the cell (representing total cellular fluorescence). The mean fluorescence intensity associated with the Golgi and total cell was measured for Arf-1-GFP. The total Arf-1-GFP fluorescence associated with the Golgi was expressed as a fraction of the total cellular fluorescence, and this procedure was repeated for 10 cells. Error bars are the mean ± SD.