anti dc sign  (R&D Systems)

Journal: International Journal of Molecular Sciences

Article Title: Identification of Important N-Linked Glycosylation Sites in the Hemagglutinin Protein and Their Functional Impact on DC-SIGN Mediated Avian Influenza H5N1 Infection

doi: 10.3390/ijms22020743

Figure Lengend Snippet: Generation of recombinant DC-SIGN and H5N1 pseduotyped virus. ( A ) The plasmid encoded extracellular carbohydrate interacting domain (ECD) of DC-SIGN with Fc tag was transfected into Freestyle HEK 293F cells and incubated in an orbital shaker incubator for a further 48 h at 37 °C, 120 rpm, and 5% CO2. The supernatants were collected and subjected to further purification by protein A beads. The total lysates and purified recombinant DS-SIGN-ECD-Fc proteins were subjected to immunoblotting using anti-human IgG (the left) or anti-DC-SIGN antibodies (the right) to check protein expression level. ( B ) Mock and pDC-SIGN ECD-Fc transfected cells were subjected to immunofluorescent staining with mouse anti-DC-SIGN antibodies followed by the addition of red fluorescent labeled antibodies against the primary antibodies. The stained cells were observed using a fluorescent microscope. ( C ) Avian H5N1 pseudotyped viruses (PVs) were generated by using co-transfection of pNL-Luc-E-R- vector with pHW1203-HA and pHW1203-NA vectors into HEK293T cells. Cell supernatant containing H5N1-PVs were collected 48 h post-transfection. After purification and concentration by ultracentrifugation, the virions were subjected to transmission electron microscope (TEM), as well as immuno-transmission electron microscope (immuno-TEM) which was stained with anti-HA (H5) antibodies, followed by the addition of gold labeled antibodies against the primary antibodies. ( D ) Sialidase pretreated Raji-DC-SIGN cell were incubated with mock or H5N1-PVs treatment at 4 °C for 2 h for viral binding and subjected to immunofluorescent staining with anti-HA (H5) and anti-DC-SIGN antibodies, followed by the addition of green (for HA) and red (for DC-SIGN) fluorescent labeled antibodies against the primary antibodies and then subjected to Carl Zeiss LSM 700 Confocal Microscope observation. ( E ) Comparison the infectivity among lentivirus pseudotyped with H5N1, H1N1, and H3N2 envelope in Raji and Raji-DC-SIGN cells. Representative results are shown. Quantitative data represent the means ± SD of results from at least three independent experiments (* p < 0.05; ** p < 0.01).

Article Snippet: For confirmation of expression of DC-SIGN, the pcDNA3/hIgG1.Fc(mut)-DC-SIGN.ECD vectors were transfected to 293-F cells and incubated at 37 °C for 48 h. The cells were subjected to immunostaining with mouse anti-DC-SIGN monoclonal antibodies (R&D, Cat. No. MAB161) (1:1000) and incubated at room temperature for 1 h. After three washes with PBS, the cells were strained with goat anti-mouse-IgG conjugated Alexa555 (Abcam, Cat. No. ab150118) (1:1000).

Techniques: Recombinant, Plasmid Preparation, Transfection, Incubation, Purification, Western Blot, Expressing, Staining, Labeling, Microscopy, Generated, Cotransfection, Concentration Assay, Transmission Assay, Binding Assay, Infection

Journal: International Journal of Molecular Sciences

Article Title: Identification of Important N-Linked Glycosylation Sites in the Hemagglutinin Protein and Their Functional Impact on DC-SIGN Mediated Avian Influenza H5N1 Infection

doi: 10.3390/ijms22020743

Figure Lengend Snippet: Evaluation of DC-SIGN mediated trans infection among H5N1-PVs carrying N-glycosylation mutations. ( A ) The scheme of modified conventional capture assay is demonstrated. ( B ) Raji and Raji-DC-SIGN were used as captured cells. They were incubated with H5N1-PVs at 4 °C for 2 h and then co-cultured with MDCK (target cells) at 37 °C for 24–48 h. After co-culturing, the capture cells were removed via intensive PBS washing (three to five times) and the target MDCK cells were subjected to luminescence analysis. In addition, for detecting the virions budding from cis infection, the transwell system was used to monitor those virions released from captured cells further causing MDCK (target cells) infection. The lower channel of infected MDCK cells in the transwell were also subjected to luminescence analysis. Alternatively, some groups were co-treated with IgG control and anti-DC-SIGN monoclonal antibodies. The relative infectivity was measured by using the luminescence values of co-cultured MDCK, normalized with values of MDCK from transwell system. ( C ) The Raji and Raji-DC-SIGN cells (captured cells) were incubated with H5N1-PVs carrying different N-glycosylation mutations on HA at 4 °C for 2 h and then co-cultured with MDCK (target cells) at 37 °C for 24–48 h. After co-culturing, the capture cells were removed via intensive PBS washing (three-five times) and the target MDCK cells were subjected to luminescence analysis. Similarly, the detection of the virions released from cis infection of the captured cells was monitored using transwell system mentioned above. Alternatively, some groups were co-treated with IgG control and anti-DC-SIGN monoclonal antibodies. The relative infectivity was measured by using the luminescence values of co-cultured MDCK, normalized with values of MDCK from transwell system. The significant difference was measured by each N-glycosylation mutant compared to WT group. Representative results are shown. Quantitative data represent the means ± SD of results from at least three independent experiments (WT, wild-type) (* p < 0.05; ** p < 0.01).

Article Snippet: For confirmation of expression of DC-SIGN, the pcDNA3/hIgG1.Fc(mut)-DC-SIGN.ECD vectors were transfected to 293-F cells and incubated at 37 °C for 48 h. The cells were subjected to immunostaining with mouse anti-DC-SIGN monoclonal antibodies (R&D, Cat. No. MAB161) (1:1000) and incubated at room temperature for 1 h. After three washes with PBS, the cells were strained with goat anti-mouse-IgG conjugated Alexa555 (Abcam, Cat. No. ab150118) (1:1000).

Techniques: Infection, Modification, Incubation, Cell Culture, Mutagenesis