Figure 4. Brefeldin A inhibits surface delivery of GPI-APs, but enhances endocytosis via the GEEC pathway.

(a) CHO cells transiently transfected with CFP–GPI were grown at 20 °C for 16 h and then shifted to 37 °C in presence (+BFA, 20 μg ml^–1) or absence of Brefeldin A (–BFA) for 1 h. Surface levels of CFP–GPI, monitored by labelling cells with anti-CFP at 4 °C, shows that BFA-treatment blocks exocytic delivery of CFP–GPI. The histogram shows anti-CFP antibody fluorescence at the surface of cells, normalized to total CFP–GPI expression per cell, and plotted as a ratio to the cell surface levels measured at 20 °C. The error bars represent weighted mean ± s.e.m. (n = 56, 40, 55). (b) IA2.2 cells, treated with BFA (20 μg ml^–1 for 1 h at 37 °C) were assayed for FR–GPI and the fluid-phase uptake as described in Fig. 1. In BFA-treated cells, fluid-phase and FR–GPI uptake is enhanced. (c) Histogram showing quantification of fluorescence of endocytosed probes (normalized to FR–GPI expression at the surface for FR-GPI uptake) in cells treated with BFA in the presence or absence of ARF1^T31N transfection, represented as the ratio of uptake to that observed in untreated cells (control). The error bars represents weighted mean ± s.e.m. (n = 98, 139, 71 for fluid and 103, 112, 64 for FR). The single and double asterisks represent P values (<0.002) from the indicated comparisons. The scale bars in a and b represent 10 μm.