Figure 5
Figure 5. Pathogen-recognition ability of LSECtin. (A) Generation of LSECtin stable transfectants. K562 cells were transfected with an empty vector (Mock) or with an LSECtin expression vector (LSECtin) and grown in G418-containing selective medium. LSECtin expression was determined by indirect immunofluorescence with a polyclonal antiserum against LSECtin (ADS-4) or the SOTO1 anti-LSECtin monoclonal antibody. Preimmune polyclonal antiserum (Preimm.) and the supernatant from the P3 × 63 myeloma (X63) were used as controls. (B) Mock-transfected K562 cells (Mock) or K562-LSECtin cells stably transfected with full-length LSECtin (LSECtin) were surface-labeled with a water-soluble and membrane-impermeable biotin derivative (EZ-linked Sulfo-NHS-LC-Biotin; Pierce, Rockford, IL), washed, lysed, and immunoprecipitated with polyclonal antisera against LSECtin (left panel, ADS-3; right panel, ADS-4) or preimmune serum. Precipitated material was resolved by SDS-PAGE under reducing conditions and subjected to Western blot with HRP-streptavidin. (C) Binding of Ebola and Marburg pseudovirus to LSECtin-transfected cells. Mock-transfected and K562-LSECtin transfectants were challenged with vesicular stomatitis virus (VSV), Marburg or Ebola virus GP pseudotypes, and cells were assayed for luciferase expression 48 hours postinfection. (D) Inhibitory effect of the ADS-1 anti-LSECtin polyclonal antiserum on the Ebola pseudovirus binding to LSECtin. The experiment was performed like in D, but cells were preincubated for 10 minutes at room temperature with the preinmune or ADS-1 polyclonal antibodies before viral addition. (E) Binding of Ebola virus GP1 and HIV gp120 to MDDC. Cells were incubated with the recombinant proteins either in the absence or in the presence of antibodies against DC-SIGN (MR1), LSECtin (ADS-1), or both and binding measured by flow cytometry. Protein binding is measured as relative fluorescence, which indicates the binding observed in each experimental condition relative to the binding detected in the presence of preimmune antiserum (considered as 100). The level of expression of LSECtin and DC-SIGN in the assayed MDDC is illustrated in the upper panel.

Pathogen-recognition ability of LSECtin. (A) Generation of LSECtin stable transfectants. K562 cells were transfected with an empty vector (Mock) or with an LSECtin expression vector (LSECtin) and grown in G418-containing selective medium. LSECtin expression was determined by indirect immunofluorescence with a polyclonal antiserum against LSECtin (ADS-4) or the SOTO1 anti-LSECtin monoclonal antibody. Preimmune polyclonal antiserum (Preimm.) and the supernatant from the P3 × 63 myeloma (X63) were used as controls. (B) Mock-transfected K562 cells (Mock) or K562-LSECtin cells stably transfected with full-length LSECtin (LSECtin) were surface-labeled with a water-soluble and membrane-impermeable biotin derivative (EZ-linked Sulfo-NHS-LC-Biotin; Pierce, Rockford, IL), washed, lysed, and immunoprecipitated with polyclonal antisera against LSECtin (left panel, ADS-3; right panel, ADS-4) or preimmune serum. Precipitated material was resolved by SDS-PAGE under reducing conditions and subjected to Western blot with HRP-streptavidin. (C) Binding of Ebola and Marburg pseudovirus to LSECtin-transfected cells. Mock-transfected and K562-LSECtin transfectants were challenged with vesicular stomatitis virus (VSV), Marburg or Ebola virus GP pseudotypes, and cells were assayed for luciferase expression 48 hours postinfection. (D) Inhibitory effect of the ADS-1 anti-LSECtin polyclonal antiserum on the Ebola pseudovirus binding to LSECtin. The experiment was performed like in D, but cells were preincubated for 10 minutes at room temperature with the preinmune or ADS-1 polyclonal antibodies before viral addition. (E) Binding of Ebola virus GP1 and HIV gp120 to MDDC. Cells were incubated with the recombinant proteins either in the absence or in the presence of antibodies against DC-SIGN (MR1), LSECtin (ADS-1), or both and binding measured by flow cytometry. Protein binding is measured as relative fluorescence, which indicates the binding observed in each experimental condition relative to the binding detected in the presence of preimmune antiserum (considered as 100). The level of expression of LSECtin and DC-SIGN in the assayed MDDC is illustrated in the upper panel.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal