Ligand-induced internalization of LSECtin in transfectants and monocyte-derived dendritic cells. (A) Monoclonal antibody-induced internalization of LSECtin in K562 transfectants. K562-LSECtin cells were incubated with either the SOTO1 (antiLSECtin) or the P5D2 (antiCD29) antibodies for 30 minutes at 4°C. After extensive washing in cold phosphate-buffered saline, cells were transferred to 37°C for the indicated time points, fixed, and the presence of cell surface-bound antibodies detected with FITC-labeled F(ab′)₂ anti-mouse IgG. Where indicated, cells were fixed and permeabilized before addition of the fluorescent secondary antibody (Perm). Values represent the fluorescence intensity of the cells at the distinct time points relative to the fluorescence intensity of control cells maintained at 4°C. One representative experiment of two is shown. (B) Ebola GP1-induced internalization of LSECtin. K562-LSECtin cells were either untreated or incubated with Ebola virus GP1-Fc (GP1) for 10 minutes at 37°C, transferred to 4°C, and the expression of LSECtin or the CD29 integrin (negative control) determined by indirect immunofluorescence with SOTO1 or P5D2 monoclonal antibodies and FITC-labeled F(ab′)2 anti-mouse IgG. Values represent the fluorescence intensity of the cells at the distinct time points relative to the fluorescence intensity of control cells maintained at 4°C. Mean ± standard deviation of three independent experiments is shown (*P = 0.001). (C) Cytoplasmic motifs involved in LSECtin ligand-induced internalization. K562 cells were transiently transfected with expression vectors for WT LSECtin or LSECtin mutated at Y⁶ (LSECtin Y/F), E¹⁴E¹⁵ (LSECtin EE/AA), W²¹GRW²⁴VHW²⁷ (LSECtin 3W/3A), or at both Y⁶ and E¹⁴E¹⁵ (LSECtin DM). Twenty-four hours later, cells were washed and incubated with the SOTO1 (anti-LSECtin) or the P5D2 (anti-CD29) antibodies for 30 minutes at 4°C. After extensive washing in cold phosphate-buffered saline, cells were transferred to 37°C for the indicated time points (2, 5, 15 minutes), placed on ice, and the presence of cell surface-bound antibodies detected with FITC-labeled F(ab′)₂ anti-mouse IgG. Values represent the fluorescence intensity of the cells at the distinct time points relative to the fluorescence intensity of control cells maintained at 4°C. Shown are the mean and standard deviation of three independent experiments. (D) Monoclonal antibody-induced internalization of LSECtin in MDDC. MDDC were incubated with either the SOTO1 (anti-LSECtin) or the TS1/18 (anti-CD18) antibodies for 30 minutes at 4°C. After extensive washing in cold phosphate-buffered saline, cells were transferred to 37°C for the indicated time points and immediately transferred to 4°C. The presence of cell surface-bound antibodies was detected with FITC-labeled F(ab′)₂ anti-mouse IgG. Relative expression of each protein was measured by multiplying the percentage of marker-positive cells by their mean fluorescence intensity and is referred to the value obtained for control cells maintained at 4°C (considered as 100). One representative experiment of two is shown.