FIGURE 2:

Knockdown of VAMP5 suppresses FcR-mediated phagocytosis but not phagosome-lysosome fusion. (A) Total cell lysates from J774 cells 72 h after transfection with a nonspecific control siRNA (si-control), VAMP5 siRNA (si-VAMP5), and SNAP23 siRNA (si-SNAP23) were analyzed by western blotting with the indicated antibodies. (B) siRNA-transfected cells were incubated with IgG-opsonized Texas Red-zymosan, and the efficiencies of association (left) and phagocytosis (right) were measured, as described in Figure 1C. Data are presented as the mean ± SEM of three (left) or four (right) independent experiments. (C) siRNA-transfected cells labeled late endosomes/lysosomes with RB-dextran and total RB fluorescence were measured (left). The percentage of RB-dextran-positive phagosomes was calculated, as described in Figure 1D (right). Data are presented as the mean ± SEM of three to six (left) or three (right) independent experiments. (D) J774/Myc and J774/Myc-VAMP5 cells transfected with the indicated siRNAs were analyzed for association and phagocytosis efficiencies. The efficiencies of association (left) and phagocytosis (right) in siRNA-transfected cells were measured, as described in Figure 1C. Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test (the decimal numbers above the bars represent the p values).