Journal: Science Advances

Article Title: Vibrio MARTX toxin binding of biantennary N-glycans at host cell surfaces

doi: 10.1126/sciadv.adt0063

Figure Lengend Snippet: ( A ) ELISA binding curve of Aknot-HA to the ectodomain of L1CAM. High-absorbance plates were coated with L1CAM across a concentration gradient, reaching up to 1 μg/ml. The binding intensities Aknot-HA were assessed across a range of specified concentrations. Data points are presented as means ± SD from n = 3 individual experiments and fitted to a one-site specific binding least-squares fit model in Prism 10. ( B ) Representative flow cytometry plots of Aknot-HA binding to HEK293T cells. Cells were transfected with either an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 100 nM Aknot-HA or PBS. The binding signal of Aknot-HA was detected by Alexa Fluor 647–conjugated anti-HA; surface expression of L1CAM was detected by phycoerythrin (PE)–conjugated anti-L1CAM. ( C ) Immunofluorescence imaging of Aknot-HA and L1CAM on nonpermeabilized HEK293T cells transfected with an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 500 nM Aknot-HA. ( D ) Correlation of L1CAM and Aknot-HA mean pixel intensity for each cell in (C) quantified from n = 3 individual experiments analyzed by CellProfiler. Simple linear regression was fitted in Prism 10, best fit was shown in solid line, and 95% confidence intervals were shown in dashed line ( R 2 = 0.85). ( E ) Quantification of flow cytometry analysis of Aknot-HA binding to control and L1CAM −/− HeLa Cas9 cells detected by Alexa Fluor 647–conjugated anti-HA antibody. The average MFI was normalized to 25 nM Aknot-HA binding to control cells for each experiment. Data are presented as means ± SD from n = 3 individual experiments (**** P < 0.0001, Student’s t tests).

Article Snippet: For transfection, 2.5 μg of plasmid DNA containing the L1CAM gene (Addgene, catalog no. 89411), CDH1-GFP (Addgene, catalog no. 28009), or the corresponding empty vector control pcDNA3 was prepared.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay, Flow Cytometry, Transfection, Plasmid Preparation, Sequencing, Incubation, Expressing, Immunofluorescence, Imaging, Control

Journal: Science Advances

Article Title: Vibrio MARTX toxin binding of biantennary N-glycans at host cell surfaces

doi: 10.1126/sciadv.adt0063

Figure Lengend Snippet: ( A ) ELISA binding curve of Aknot-HA to the ectodomain of L1CAM treated with and without PNGaseF. Plates were coated with L1CAM across a concentration gradient. The binding intensities of Aknot-HA were assessed at 1 μM. Means ± SD from n = 3 individual experiments fitted to a one-site specific binding least-squares fit model. The N-glycosylation sites on the L1CAM ectodomain are indicated as red straight lines, with the yellow box indicating the cell membrane. ( B ) Flow cytometry analysis of 100 nM Aknot-HA binding to HEK293T and GNT1 −/− cells. Means ± SD from n = 3 individual experiments (*** P < 0.001, Student’s t test). ( C ) Flow cytometry analysis of Aknot-HA binding to HEK293T and GNT1 −/− cells. Both cell lines were transfected with either an empty vector (pcDNA3.1) or the vector encoding the L1CAM sequence. Forty-eight hours after transfections, cells were incubated with 100 nM Aknot-HA or PBS. The binding signal of Aknot-HA was detected by Alexa Fluor 647–conjugated anti-HA. Means ± SD from n = 3 individual experiments (*** P < 0.001, Student’s t test). ( D ) Flow cytometry analysis of 100 nM Aknot-HA binding to L1CAM −/− HeLa Cas9 cells treated with or without kifunensine at 37°C overnight. Means ± SD from n = 3 individual experiments (**** P < 0.0001, Student’s t test). ( E ) Representative flow cytometry plots of Aknot-HA binding to HEK293T cells. Cells were transfected with either an empty pcDNA3.1 vector (Vehicle) or the vector carrying the gene fusion CDH1 - GFP , which encodes green fluorescent protein (GFP)–tagged E-cadherin (E-CAD). Forty-eight hours after transfections, cells were incubated with 100 nM Aknot-HA. The binding signal of Aknot-HA was detected by the APC channel. The ectopic expression of GFP-tagged E-cadherin was detected by the fluorescein isothiocyanate channel. The cartoon on the left illustrates the N-glycosylation sites (red straight lines) on the E-cadherin ectodomain, with the yellow box indicating the cell membrane.

Article Snippet: For transfection, 2.5 μg of plasmid DNA containing the L1CAM gene (Addgene, catalog no. 89411), CDH1-GFP (Addgene, catalog no. 28009), or the corresponding empty vector control pcDNA3 was prepared.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay, Glycoproteomics, Membrane, Flow Cytometry, Transfection, Plasmid Preparation, Sequencing, Incubation, Expressing