Journal: Immunity

Article Title: Profiling B cell immunodominance after SARS-CoV-2 infection reveals antibody evolution to non-neutralizing viral targets

doi: 10.1016/j.immuni.2021.05.001

Figure Lengend Snippet:

Article Snippet: Goat anti-human IgG-biotin , Mabtech , Cat# 3820-4-250.

Techniques: Virus, Clinical Proteomics, Infection, Recombinant, RNA Binding Assay, Transfection, Enzyme-linked Immunosorbent Assay, Bioprocessing, Multiplex Assay, Isolation, Gene Expression, Plasmid Preparation, Software

Journal: PLoS ONE

Article Title: The bioactivity of soluble Fas ligand is modulated by key amino acids of its stalk region

doi: 10.1371/journal.pone.0253260

Figure Lengend Snippet: (A) Competition assay. Serial 3-fold dilutions of either WT or 8-site mutated sFasL were mixed with biotinylated WT sFasL (70 ng/ml) and incubated for 2 hours in 96-well plates pre-coated with recombinant human soluble Fas (250 ng/ml). Biotin was detected using HRP conjugated streptavidin; (B) Fas binding assay. Serial 3-fold dilutions of wild type (WT) or mut-sFasL were incubated for 2 hours in wells coated with recombinant human soluble Fas, 5 μg/ml. The sFasL bound to Fas was detected using a polyclonal anti-human FasL antibody. The mut sFasL binding curve was shifted to the left. n = 3. (C) Jurkat cells were incubated in 96-well plates at a concentration of 1 x 10 4 cells/well. Different molar ratios of the Fas-activating antibody CH-11 and mut-sFasL were added, and caspase 3/7 activity measured after 5 hours. The mut-sFasL did not inhibit the activity of CH11. Data represent the results from three separate experiments, each done in duplicate, and were analyzed by 2-way ANOVA with Sidak’s post hoc analysis; (A), WT-sFasL compared to mut-sFasL at the same concentrations; (B), comparisons made to no competitor condition for each molecule; (C) Comparisons made to unmixed antibody (Ab). Each dot represents individual data, lines represent means ± SD. * = P < 0.05; ** = P<0.01, ***P<0.001.

Article Snippet: For the Fas-FasL binding assay, we used recombinant human soluble Fas receptors (PeproTech, Rocky Hill, NJ) and biotinylated goat anti-human FasL polyclonal antibodies (PeproTech Cat# 500-P184bt-50ug, RRID:AB_148184).

Techniques: Competitive Binding Assay, Incubation, Recombinant, Binding Assay, Concentration Assay, Activity Assay

Journal: PLoS ONE

Article Title: The bioactivity of soluble Fas ligand is modulated by key amino acids of its stalk region

doi: 10.1371/journal.pone.0253260

Figure Lengend Snippet: Serial 3-fold dilutions of FLAG tagged mut-sFasL were mixed with biotinylated WT sFasL (60 ng). The mixtures were incubated at room temperature for 15 minutes and applied to streptavidin-coated wells. FLAG tags were detected with HRP anti-FLAG mAb. There was a dose-dependent increase in FLAG signal in wells incubated with the biotinylated WT sFasL and the mut-sFasL-FLAG mixture, indicating formation of complexes of WT sFasL and mut-sFasL. The mut-sFasL-FLAG without biotin did not bind to the streptavidin-coated plates. Data shown as ratio of the absorbance at 450 nm of each experimental condition to that of media only. Data were generated from three separate experiments, each done in duplicate, and were analyzed by 2-way ANOVA with Dunnet’s post hoc analysis; comparisons were made to 0 ng/mL for each condition. Each dot represents individual data, lines represent means ± SD. * = P < 0.05; ** = P<0.01, ***P<0.001.

Article Snippet: For the Fas-FasL binding assay, we used recombinant human soluble Fas receptors (PeproTech, Rocky Hill, NJ) and biotinylated goat anti-human FasL polyclonal antibodies (PeproTech Cat# 500-P184bt-50ug, RRID:AB_148184).

Techniques: Incubation, Generated