Journal: The Journal of Clinical Investigation
Article Title: Thrombospondin-1 inhibits alternative complement pathway activation in antineutrophil cytoplasmic antibody-associated vasculitis
doi: 10.1172/JCI180062
Figure Lengend Snippet: ( A ) TSP-1 protects FB from cleavage by FD. FB was incubated with FD, C3b, and varying concentrations of TSP-1. Subsequently, FB and its cleavage products were visualized by Coomassie blue staining. Graph on the right illustrates band intensity of FB and its cleavage products. ( B ) TSP-1 inhibits cleavage of C3 by the alternative complement pathway C3 convertase. C3 convertase was generated by incubating C3(H2O) with FB and FD, followed by the addition of C3 in the presence or absence of TSP-1 or FH. As a positive control, CVF C3 convertase (CVF + FB + FD) was utilized. The graph on the right depicts the band intensity of C3-α′ chain. Lanes were run on the same gel but were noncontiguous. Data are shown as mean ± SD. ( C ) TSP-1 inhibits cleavage of C5. Cobra venom factor (CVF) convertase (CVFBb) was generated and added to C5 preincubated with FH, eculizumab, MFHR1, or TSP-1. The amount of released C5a was quantified by ELISA. Data are shown as mean ± SD. ANOVA was used with Dunnett’s multiple-comparison test. ( D ) TSP-1 inhibits the formation of MAC. Sheep erythrocytes were premixed with C7 (9 nM), C8 (7 nM), and C9 (15 nM). BSA, eculizumab, MFHR1, or TSP-1 (1.3 μM each) were preincubated with C5b-6 (0.7 nM) and then added to the erythrocytes. Data are shown as mean ± SD of 3 independent experiments; ** P ≤ 0.01, *** P ≤ 0.01; 1-way ANOVA was used with Tukey’s post hoc test for comparison against control.
Article Snippet: The following ELISAs and sample dilutions were used in this study: mouse albumin ELISA kit (1:500.000, Crystal Chem, 80630), mouse creatinine (Cr) ELISA kit (1:10, AFG Scientific, EK734005 ), mouse complement C3 ELISA kit (1:300, Abcam, AB157711 ), mouse Cit H3 ELISA kit (1:10, Biorbyt, ORB1496437), mouse C5a ELISA kit (1:25, Antikoerper Online, ABIN6720057), and mouse TSP-1 (THBS1) ELISA kit (1:60, Biozol, ABX575980).
Techniques: Incubation, Staining, Generated, Positive Control, Combined Bisulfite Restriction Analysis Assay, Enzyme-linked Immunosorbent Assay, Comparison, Control
