American Society of Hematology

Figure 3

$Figure 3. Expression of LSECtin in human MDDC. (A) Specificity of polyclonal antisera against LSECtin by enzyme-linked immunosorbent assay. High protein-binding 96-well plates were coated with either purified HIS-LSECtin, purified βgal-HIS, or BSA. After washing, wells were treated with either a monoclonal antibody against the HIS epitope (anti-His), a control monoclonal antibody (antiCD86), polyclonal antisera against LSECtin (ADS-1, ADS-3, ADS-4), or the corresponding preimmune sera. Bound antibodies were detected using HRP-conjugated goat antirabbit and goat antimouse polyclonal antisera. Quantification was done on a microplate enzyme-linked immunosorbent assay reader at 450 nm. (B) Specificity of polyclonal antisera against LSECtin by Western blot. Whole cell extracts were obtained from COS-7 cells transiently transfected with full-length LSECtin, the Δ2 LSECtin isoform (LSECtin Δ2), or mock-transfected cells (Mock). Ten micrograms of each whole cell extract was subjected to Western blot using the ADS-1 or ADS-4 polyclonal antisera specific for LSECtin. (C) Whole cell lysates were obtained from immature and mature MDDC, and 10 μg of each extract was subjected to Western blot using the ADS-1 anti-LSECtin polyclonal antiserum either alone (left panel) or in the presence of an excess of the immunizing peptide (right panel). Lysates from mock- and LSECtin-transfected COS-7 cells were included as controls. The position of the LSECtin protein is indicated by an arrowhead, and nonspecific bands are denoted as NS. (D) Immature MDDC were lysed in 1% Brij 98 lysis buffer at 37°C and fractionated by sucrose density gradient centrifugation. The low-density Brij 98-insoluble fractions 2-4 (lanes 2-4, Rafts) and the high-density Brij 98-soluble fractions 6-8 (lanes 6-8, soluble) were separated by 12.5% SDS-PAGE under reducing conditions, and the distribution of LSECtin was determined by immunoblotting. Cytoskeletal-associated Rafts (CAR), obtained by solubilization of the cell pellet with Brij98 + Octyl D-glucoside in lysis buffer, were analyzed in parallel (lane 9, CAR). Ten micrograms of each fraction was subjected to Western blot, and the distribution of LSECtin and ganglioside GM1 in the distinct fractions was determined by immunoblotting with the ADS-1 polyclonal antisera (LSECtin) or cholera toxin-HRP (GM1). The experiment was done on MDDC from two independent donors, and one of them is shown.$

Expression of LSECtin in human MDDC. (A) Specificity of polyclonal antisera against LSECtin by enzyme-linked immunosorbent assay. High protein-binding 96-well plates were coated with either purified HIS-LSECtin, purified βgal-HIS, or BSA. After washing, wells were treated with either a monoclonal antibody against the HIS epitope (anti-His), a control monoclonal antibody (antiCD86), polyclonal antisera against LSECtin (ADS-1, ADS-3, ADS-4), or the corresponding preimmune sera. Bound antibodies were detected using HRP-conjugated goat antirabbit and goat antimouse polyclonal antisera. Quantification was done on a microplate enzyme-linked immunosorbent assay reader at 450 nm. (B) Specificity of polyclonal antisera against LSECtin by Western blot. Whole cell extracts were obtained from COS-7 cells transiently transfected with full-length LSECtin, the Δ2 LSECtin isoform (LSECtin Δ2), or mock-transfected cells (Mock). Ten micrograms of each whole cell extract was subjected to Western blot using the ADS-1 or ADS-4 polyclonal antisera specific for LSECtin. (C) Whole cell lysates were obtained from immature and mature MDDC, and 10 μg of each extract was subjected to Western blot using the ADS-1 anti-LSECtin polyclonal antiserum either alone (left panel) or in the presence of an excess of the immunizing peptide (right panel). Lysates from mock- and LSECtin-transfected COS-7 cells were included as controls. The position of the LSECtin protein is indicated by an arrowhead, and nonspecific bands are denoted as NS. (D) Immature MDDC were lysed in 1% Brij 98 lysis buffer at 37°C and fractionated by sucrose density gradient centrifugation. The low-density Brij 98-insoluble fractions 2-4 (lanes 2-4, Rafts) and the high-density Brij 98-soluble fractions 6-8 (lanes 6-8, soluble) were separated by 12.5% SDS-PAGE under reducing conditions, and the distribution of LSECtin was determined by immunoblotting. Cytoskeletal-associated Rafts (CAR), obtained by solubilization of the cell pellet with Brij98 + Octyl D-glucoside in lysis buffer, were analyzed in parallel (lane 9, CAR). Ten micrograms of each fraction was subjected to Western blot, and the distribution of LSECtin and ganglioside GM1 in the distinct fractions was determined by immunoblotting with the ADS-1 polyclonal antisera (LSECtin) or cholera toxin-HRP (GM1). The experiment was done on MDDC from two independent donors, and one of them is shown.

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