First, we examined whether FITC-FSL-1 is able to activate macrophages, because there is a possibility that FITC conjugation affects the ability of FSL-1 to activate them.13 It was found that both FITC-FSL-1 and FSL-1 induced tumour necrosis factor-α production by a murine macrophage cell line, RAW264.7 cells, in a dose-dependent manner (Fig. 1), suggesting that FITC-FSL-1 is also able to activate macrophages, possibly through TLR2. However, it remains unknown

how FSL-1 is processed in macrophages after recognition by TLR2. To address this question, an experiment was carried out to determine whether FSL-1 is internalized by macrophages after recognition by TLR2. RAW264.7 cells were incubated with FITC-FSL-1 for 2 hr at 4° (on ice) or at 37°, and then uptake of FSL-1 was determined. FSL-1 was found in the cell membrane but not in the cytosol of RAW264.7 Selinexor in vitro cells at 4° (Fig. 2a,c). However, FSL-1 was found in both the cell membrane find protocol and the

cytosol of the cells at 37° (Fig. 2b,d). These results suggest that FSL-1 is clearly internalized into the cells in a temperature-dependent manner. To confirm whether FSL-1 is specifically internalized, effects of unlabelled FSL-1 on FITC-FSL-1 uptake were also examined. It was found that unlabelled FSL-1 significantly inhibited FITC-FSL-1 in a dose-dependent manner (Fig. 3), suggesting that FITC-FSL-1 uptake by aminophylline the cells occurs specifically. It has been demonstrated that modes of endocytosis of small soluble

molecules can be mainly divided into three pathways: clathrin-, caveolae- and lipid raft-dependent endocytic pathways.17 Therefore, experiments were carried out to determine the effects of three chemicals, Nys, CPZ and MbCD, on internalization of FSL-1. Nys selectively disrupts caveolae- and lipid raft-dependent endocytosis but has no effect on clathrin-dependent endocytosis.18 CPZ disrupts clathrin-dependent endocytosis.19 MbCD disrupts both lipid raft- and clathrin-dependent endocytosis.20–23 It has been demonstrated that TLR2 is enriched in lipid rafts24 and that the TLR2 ligand LTA is internalized into cells with TLR2 via lipid rafts.15 The present study demonstrated that Nys had no effect on FSL-1 uptake by RAW264.7 cells (Fig. 4a–c), suggesting that FSL-1 is not internalized by caveolae- and lipid raft-dependent endocytosis. Both CPZ and MbCD inhibited FSL-1 uptake by the cells in a dose-dependent manner (Fig. 4d–i), suggesting that FSL-1 is internalized into macrophages by a clathrin-dependent endocytosis. The next experiment was therefore carried out to determine whether FSL-1 is co-localized with clathrin in cells. RAW264.7 cells were incubated for 2 hr with FITC-FSL-1, permeabilized with Cytofix/Cytoperm (BD Biosciences), and treated with an anti-clathrin mAb.