Fig. 4

eCIRP downregulates Rac1 protein levels. RAW 264.7 cells were incubated with 0.1, 0.2 and 1 µg/ml rmCIRP for 24 h (A) or with 1 µg/ml rmCIRP for 1, 5 and 24 h (B). Rac1 protein expression was assessed by Western blotting. Representative blots are shown. Data are expressed as the mean ± SD (n = 5–9/group). The PBS control was set to 1 for normalization. The groups were compared by one-way ANOVA and the SNK method. *p < 0.05 vs. PBS. C RAW 264.7 cells were incubated with rmCIRP (1 µg/ml) or PBS for 24 h. In an additional group, RAW 264.7 cells were incubated with the Rac1 inhibitor NSC23766 (30 µM) 15 min before adding pHrodo-labeled E. coli. The efficiency of phagocytosis was measured using a fluorescence plate reader. The cells were fixed at the end of the phagocytosis assay for microscopy imaging. Representative images are shown. The experiment was performed twice. Data are expressed as the mean ± SD (n = 4–5/group). The groups were compared by one-way ANOVA and the SNK method. *p < 0.05 vs. PBS. D RAW 264.7 cells were incubated with rmCIRP (1 µg/ml) or PBS for 24 h. Then, the cells were fixed for Rac1 immunofluorescence and F-actin staining. Rac1 (red), F-actin (green), and merged staining images are shown. Framed areas are enlarged to show details. The nucleus was stained with DAPI (blue). Scale bar = 50 µm