Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: A) Schematic of Factor I and cofactor cleavage of C3b and iC3b. B) Factor I cleavage assay of C3b. C3b and Factor I (FI) were incubated alone, with Factor H (FH), or C4BP, or with concentrations of Sez6L2-MH ranging from 1 to 10 μg/mL for 2 hours at 37°C. Then samples were analyzed by western blot using antibodies that recognize C3d, a region within the C3α chain (and highlighted by the black rectangle in the schematics in (A)). Coomassie stained gels are also shown. FH and C4BP are known co-factors of FI towards C3b and served as positive controls. Incubation of C3b and FI with Sez6L2-MH also generated the C3 cleavage products α’1 and α’2 showing Sez6L2-MH is a cofactor for Factor I cleavage of C3b. C) Schematic of Factor I + cofactor cleavage of C4b. D) C4b and Factor I (FI) were incubated alone, with FH, C4BP, or with concentrations of Sez6L2-MH ranging from 1 to 10 μg/mL for 2 hours at 37°C. Then samples were analyzed by western blot using a C4 polyclonal antibody or coomassie stained gels. C4 components recognized by the C4 antibody are colored black in the schematic in C. C4BP is a known cofactor of FI for C4b cleavage and served as a positive control. Incubation of C4b and FI with Sez6L2-MH did not result in the appearance of C4b cleavage products.

Article Snippet: Residual Bb on the plate was detected using polyclonal goat anti-Factor B (Comptech, A235, 1:4,000) for 1 hour at room temperature.

Techniques: Cleavage Assay, Incubation, Western Blot, Staining, Generated, Positive Control

Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: A and B) Alternative C3 convertase assay: A 96 well plate coated with C3b was incubated with Factor B and Factor D to form the C3 convertase C3bBb, then incubated with Sez6L2-MH or FH at concentrations ranging from 0 to 500 μg/mL to assess their decay accelerating activity. Factor B remaining bound to the plate (as C3bBb) was detected using an anti-Factor B antibody ELISA in (A) and Bb released into the supernatant is shown via western blot (B). C) Classical C3 convertase assay: A plate coated with C4b was incubated with C2 and C1s-enzyme to form the classical/lectin pathway C3 convertase C4b2a, then incubated with Sez6L2-MH or H-DAF at concentrations ranging from 0 to 500 μg/mL to assess their decay accelerating activity. C2 remaining bound to the plate (presumably as C4b2a) was detected using an anti-C2 antibody ELISA. For A and C ELISAs: N=3 (1 experiment with 3 replicates; representative of 2-3 independent experiments). Statistics: 1-way ANOVAS with Holms-Sidak multiple comparison’s tests were performed for each complement inhibitor with comparisons to 0 ug/mL controls. * p<0.05.

Article Snippet: Residual Bb on the plate was detected using polyclonal goat anti-Factor B (Comptech, A235, 1:4,000) for 1 hour at room temperature.

Techniques: Convertase Assay, Incubation, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Cells

Article Title: Hypoxic Processes Induce Complement Activation via Classical Pathway in Porcine Neuroretinas

doi: 10.3390/cells10123575

Figure Lengend Snippet: Primary and secondary antibodies used for immunofluorescence.

Article Snippet: Anti-factor B , Goat , Tecomedial , 1:1000 , Donkey anti-goat Alexa Fluor 488 , Dianova , 1:500.

Techniques: Immunofluorescence

Journal: Scientific Reports

Article Title: S100B immunization triggers NFκB and complement activation in an autoimmune glaucoma model

doi: 10.1038/s41598-018-28183-6

Figure Lengend Snippet: Analyses of mRNA levels via quantitative real-time PCR. Significant values are marked in bold.

Article Snippet: Anti-factor B , TECOmedical , Retina , 1:1000 , Donkey anti-goat Cy3 , Abcam , Retina , 1:600.

Techniques: Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: S100B immunization triggers NFκB and complement activation in an autoimmune glaucoma model

doi: 10.1038/s41598-018-28183-6

Figure Lengend Snippet: Activation through the lectin pathway. (A) Retinas were labeled with an antibody against factor B (red) 3, 7, and 14 days after immunization. Cell nuclei were stained with DAPI (blue). (B) No alterations in factor B + cell numbers were noted in S100 animals compared to controls at all points in time (p > 0.05). (C) The qRT-PCR analyses revealed an upregulation of factor b mRNA at 3 days (p = 0.02). No changes were observed at 7 and 14 days (p > 0.05). (D) Retinal cross-sections were stained with anti-MBL (green) and DAPI (blue) at 3, 7, and 14 days. (E) At 3 days, significantly more MBL + cells were observed in S100 retinas (p < 0.0001). No changes were noted later on (p > 0.05). (F) Expression levels of Mbl measured with qRT-PCR at 3, 7, and 14 days. At 3 days, a significant increase of Mbl mRNA expression was detected in S100 retinas (p = 0.048). Mbl went back to control level later on (p > 0.05). (G) Optic nerve sections were labeled with an anti-MBL antibody (green) and cell nuclei with DAPI (blue) 3, 7, and 14 days after immunization. (H) The number of MBL + cells remained unaltered in S100 optic nerves at 3 days (p > 0.05). After 7 days, significantly more MBL + cells were noted in the S100 group (p = 0.03) and were still present at 14 days (p = 0.02). Abbreviation: GCL = ganglion cell layer. Values are mean ± SEM for immunohistology and median ± quartile + maximum/minimum for qRT-PCR. Scale bars: 20 µm.

Article Snippet: Anti-factor B , TECOmedical , Retina , 1:1000 , Donkey anti-goat Cy3 , Abcam , Retina , 1:600.

Techniques: Activation Assay, Labeling, Staining, Quantitative RT-PCR, Expressing

Journal: Scientific Reports

Article Title: S100B immunization triggers NFκB and complement activation in an autoimmune glaucoma model

doi: 10.1038/s41598-018-28183-6

Figure Lengend Snippet: Oligonucleotide sequences.

Article Snippet: Anti-factor B , TECOmedical , Retina , 1:1000 , Donkey anti-goat Cy3 , Abcam , Retina , 1:600.

Techniques: Amplification

Journal: medRxiv

Article Title: Multi-organ complement deposition in COVID-19 patients

doi: 10.1101/2021.01.07.21249116

Figure Lengend Snippet: The panel shows representative immunofluorescence images obtained from the analysis of several sections of lung autopsy samples from 9 SARS-CoV-2-positive cases. The sections were stained to reveal the presence of spike protein, IgG, MBL, C1q, C4, C3, factor B (FB) and C5b-9 as explained in materials and methods and examined by 3 independent observers. Original magnification 200x.

Article Snippet: Tissue sections of seven µm were stained with the following primary antibodies (5μg/ml): goat anti-human IgG (Sigma-Aldrich, Milan, Italy), goat anti-C1q and C4 (The Binding Site, Birmingham, UK), goat anti-C3 (Quidel, San Diego, CA, USA), and anti-Factor B (Cytotech, San Diego, CA, USA); rabbit anti-MBL (Sigma-Aldrich) and anti-SARS-CoV-2 Spike S2 (Sino Biological, Beijing, China); murine monoclonal antibody against C9 neo-antigen (aE11, kindly provided by prof. T.E.

Techniques: Immunofluorescence, Staining