Journal:

Article Title: Functional basis for complement evasion by staphylococcal superantigen-like 7

doi: 10.1111/j.1462-5822.2010.01486.x

Figure Lengend Snippet: SSL7 inhibits generation of C5a during staphylococcal opsonisation. (A,B) Staphylococci were treated with 10% human serum for 30 minutes at 37°C in the presence of 0 – 450 nM SSL7 or SSL10. In one case (“SSL7 after”), 450 nM SSL7 was added to serum supernate after opsonisation. C5a generation was measured by using supernates as stimuli for calcium mobilization in U937-C5aR cells. Results depict in (A) representative calcium mobilization plots and in (B) mean values ± SEM of three independent experiments and are expressed relative to cells treated with supernate without staphylococcal proteins. In (A), “background” and “control” represent cells stimulated with untreated serum and serum after opsonisation without SSL7, respectively. (C) Cell-bound C5 convertases were constructed on staphylococci by initial deposition of C3b and subsequent addition of soluble fD, fB, and properdin. C5 was subsequently added in the presence of 0 – 450 nM SSL7, and C5a generation in the absence of IgA was analysed by using supernates as stimuli for calcium mobilization in U937-C5aR cells. Results represent mean values ± SEM of three independent experiments and are expressed relative to cells treated with supernate without SSL7.

Article Snippet: Bound C5 was detected using anti-C5/C5a (Hycult biotechnology, The Netherlands) and peroxidase (PO)-conjugated goat anti–mouse IgG.

Techniques: Construct

Journal:

Article Title: Functional basis for complement evasion by staphylococcal superantigen-like 7

doi: 10.1111/j.1462-5822.2010.01486.x

Figure Lengend Snippet: SSL7 effects are not dependent on IgA binding. (A-C) ELISA experiments showing binding of IgA1 (A), IgA2 (B), or C5 (C) to wells coated with 450 nM SSL7 and SSL7-IgA mutant. Results represent mean values ± SEM of two independent experiments. (D) Staphylococci were opsonized with 10% human serum in the presence of 450 nM SSL7 or SSL7-IgA mutant. Supernates were examined for generated C5a by Western blotting. Representative of three experiments.

Article Snippet: Bound C5 was detected using anti-C5/C5a (Hycult biotechnology, The Netherlands) and peroxidase (PO)-conjugated goat anti–mouse IgG.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Mutagenesis, Generated, Western Blot