purified human c3a a118  (Comtech International Inc)

Journal: Science Immunology

Article Title: SARS-CoV-2 drives JAK1/2-dependent local complement hyperactivation

doi: 10.1126/sciimmunol.abg0833

Figure Lengend Snippet: ( A-D ) Confocal images ( A and C ) and quantification ( B and D ) from n =2 independent experiments showing expression of C3a and SARS-CoV-2 N-protein in SARS-CoV-2-treated or mock-infected Calu-3 cells ( A and B ) or induced pleuripotent stem cell-derived alveolar epithelial type 2 cells (iAEC2s) ( C and D ). Scale bars in A and C indicates 100m. Cell numbers are indicated below each violin and median values denoted by dots in B and D . ( E-F ) correlation between SARS-CoV-2 N-protein intensity and C3a intensity on a per cell basis in Calu-3 cells ( E ) and iAEC2s ( F ). Indicated are Pearson correlation coefficients and associated p-values. Infected and uninfected cells in ( B-D ) have been distinguished by red and blue fills, respectively. ****p<0.0001 by ANOVA.

Article Snippet: A C3a standard curve was constructed using purified human C3a (ComTech, A118) in concentrations ranging from 10nM to 0.00977nM.

Techniques: Expressing, Infection, Derivative Assay

Journal: Science Immunology

Article Title: SARS-CoV-2 drives JAK1/2-dependent local complement hyperactivation

doi: 10.1126/sciimmunol.abg0833

Figure Lengend Snippet: ( A ) chemoproteomic profiling of CFBi identified complement factor as the only target. Shown is the dose-dependent reduction of bead-binding of complement factor B from protein extracts of cells. Shown are mean and s.d. from 3 independent experiments. ( B) C3a ELISA in plasma treated with zymosan (an alternative complement pathway activator) in the presence of increasing concentrations of EDTA (a chelator of divalent cations, which stops convertase activity), a CFB blocking antibody or isotype control, the chemical CFBi or its carrier, DMSO. Bars show mean + sem; dots represent individual experiments. ( C ) confocal images (left) and quantifications (right) showing generation of C3a in mock- or SARS-CoV-2-infected iAEC2s treated with CFBi, ruxolitinib or a combination of ruxolitinib and remdesivir. Scale bar indicates 100m. Data are from n =2 independent experiments; 18191 + 660 (mean + sd) cells per condition. Bars indicate mean + sd ( A ) or sem ( B-C ). *p<0.05, ***p<0.001, ***p<0.0001 by ANOVA.

Article Snippet: A C3a standard curve was constructed using purified human C3a (ComTech, A118) in concentrations ranging from 10nM to 0.00977nM.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Activity Assay, Blocking Assay, Control, Infection

Journal: Science Immunology

Article Title: SARS-CoV-2 drives JAK1/2-dependent local complement hyperactivation

doi: 10.1126/sciimmunol.abg0833

Figure Lengend Snippet: Schematic model of SARS-CoV-2-induction of complement in respiratory epithelial cells . SARS-CoV-2 infects respiratory epithelial cells and induces an interferon response. IFNs signal via the IFN receptor to activate STAT1 via JAK1/2. STAT1 co-operates with RELA to induce transcription of IL6 and complement genes including C3 , CFB , C1R and C1S . CFB acts as an alternative pathway C3 convertase to cleave C3 intracellularly to C3a and C3b. C3a engages C3aR and C3b engages CD46 on leukocyte subsets in the lungs to drive inflammation. These events can be pharmacologically targeted with antivirals (e.g., remdesivir), JAK-STAT inhibitors (e.g., ruxolitinib) and/or cell permeable complement inhibitors, including CFBi.

Article Snippet: A C3a standard curve was constructed using purified human C3a (ComTech, A118) in concentrations ranging from 10nM to 0.00977nM.

Techniques: