FIG. 2.

Effect of dominant-negative Rab5 on fluid phase uptake and EEA1 acquisition. CHO-IIA cells were transiently transfected with dominant-negative Rab5-GFP (DN-Rab5). (A and B) The cells were incubated for 30 min in medium containing rhodamine-dextran (100 μg/ml) to assess fluid phase intake. (C and D) The cells were allowed to internalize IgG-opsonized beads for 20 min, and this was followed by fixation and EEA1 immunostaining. (A and C) GFP fluorescence; (B) fluorescence of rhodamine-dextran; (D) distribution of EEA1. Asterisks identify transfected cells. Arrows point to phagosomes. In panel B, transfected cells are outlined to facilitate identification of their boundaries. Scale bar = 10 μm. (E) Quantification of the effects of DN-Rab5 and wortmannin (Wort) on EEA1 acquisition by phagosomes in CHO-IIA cells. Cells were allowed to internalize beads for 20 min, fixed, and immunostained for EEA1. Where indicated, cells were pretreated with 100 nM wortmannin for 30 min. The cells were analyzed microscopically, and phagosomes lined by clearly observable EEA1 were scored. Data are means + SE (error bars) of four separate experiments, each with at least 100 individual phagosomes from 30 to 40 different cells counted per condition.