Figure 1.
ARF6 is activated during phagocytosis. RAW264.7 macrophages transiently expressing ARF6-WT or ARF6-T27N were incubated with IgG-SRBC for 60 min at 37°C. The cells were fixed, and external SRBCs were stained with Cy3-coupled anti–rabbit IgG antibodies. After permeabilization, the expressed HA-tagged ARF6 constructs were detected by immunofluorescence. The efficiency of association (A) or phagocytosis (B) was calculated as indicated in Materials and methods. The mean ± SEM of three independent experiments is plotted. NT, non transfected. (C) ARF6 is transiently activated during phagocytosis. RAW264.7 macrophages were incubated with medium (control) for 10 min or with IgG-SRBC for various times at 37°C. Lysates were prepared and incubated with GST-GGA31–226 (top) or GST alone (middle). The bottom panel shows aliquots of total lysates. Western blot was performed with anti-ARF6 antibodies. Data are representative of three experiments. (D) Kinetics of activation of Rac and Cdc42 during phagocytosis. RAW264.7 macrophages were activated as described in C, and lysates were prepared and incubated with GST-CRIB. Western blot was performed with anti-Rac antibodies (top), then with anti-Cdc42 (bottom). Data are representative of four experiments.