Figure 6.
Cdc42 recruits FRLα to the phagocytic cup during Fc-γ receptor–mediated phagocytosis. (A) Western blot of lysates prepared from cells transfected with siRNAs directed against FRLα or GFP (control) and probed with anti-FRLα or anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH; loading control) antibodies. (B) Phagocytic index (number of RBCs/100 macrophages) of siRNA-transfected RAW macrophages. Experiments were performed in duplicate and repeated three times. Data are the means based on counting at least 300 macrophages per experiment. (C) FRLα-GFP–expressing cell undergoing Fc-γ receptor–mediated phagocytosis. The zero time point is a reference for when the phagosome closes around the RBC being engulfed. The top panels are pseudocolored to represent the GFP/mRFP ratio at each pixel in the cell. Low ratios are represented by blue or cool colors, whereas higher ratios are represented by increasingly red or warmer colors. Bottom panels are the corresponding DIC images for each time point. The ingested RBCs are indicated by arrows. The insets depict additional magnification of the phagocytic cup. (D) Time course of formin accumulation during Fc-γ receptor–mediated phagocytosis. n indicates the number of phagocytic events analyzed for each construct. For each time point, the GFP/mRFP ratio at the cell surface in contact with the RBC (Rp) was divided by the ratio in the cell cytoplasm (Rc). Error bars represent SEM.