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: Communications Biology

Article Title: A synthetic protein as efficient multitarget regulator against complement over-activation

doi: 10.1038/s42003-022-03094-5

Figure Lengend Snippet: a C3b binding was evaluated by ELISA. Nonlinear regression using the sigmoidal equation (dose–response curve, 4 parameters) and comparison of IC 50 showed no significant differences between MFHR13 and MFHR I62 binding to C3b ( P = 0.8426). Data represent mean values ± SD from 5 independent experiments. Absorbance values from one representative experiment are shown in Supplementary Fig. . b Cofactor activity of FI-mediated proteolytic cleavage of C3b α′-chain in the presence of MFHR1 I62 , FH, or MFHR13, evaluated in increasing reaction times. Densitometric analysis of C3b cleavage. The amount of intact α′-chain was normalized for each sample with the β-chain and set to 100% intact C3b α′-chain at time zero. The data represent mean value from 5 or 6 independent experiments ± SD. c One representative experiment included in b is shown. C3b, FI, and MFHR1 I62 , MFHR13 or FH were incubated for 10 and 20 min and C3b cleavage was visualized by SDS-PAGE and Coomassie staining. d MFHR13 displaced C3 convertases more efficiently than MFHR1 I62 and MFHR1 V62 . C3bBb were assembled in C3b-coated microtiter plates with FB and FD. Complement regulators were added and the dissociation of the C3 convertases was detected by the amount of FB using polyclonal anti-FB antibodies. Absorbance of samples without regulator was set to 100% FB and samples without FD as the negative control. Nonlinear regression using the sigmoidal equation and comparison of IC 50 indicated significant differences in DAA between MFHR13, MFHR1 V62 , and MFHR1 I62 ( P < 0.0001 for each comparison) and not significant difference between MFHR13 and hFH ( P = 0.2022). The data represent mean value from 2 independent measurements ± SD.

Article Snippet: The Bb fragments that remain bound to C3b were detected by an anti-factor B polyclonal antibody (Merck, Darmstadt, Germany), followed by HRP-conjugated rabbit anti-goat (Dako, Hamburg, Germany).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Comparison, Activity Assay, Incubation, SDS Page, Staining, Negative Control

Journal: Communications Biology

Article Title: A synthetic protein as efficient multitarget regulator against complement over-activation

doi: 10.1038/s42003-022-03094-5

Figure Lengend Snippet: a C5 binding was measured by ELISA. Nonlinear regression using the sigmoidal equation and comparison of IC 50 showed similar binding profiles for MFHR13 and MFHR1 I62 to C5 ( P = 0.2565). Data represent mean values ± SD from 4 or 5 independent experiments. Absorbance values from one representative experiment are shown in Supplementary Fig. . b MFHR1 binds to the individual TCC proteins; hFH does not. MFHR1 or hFH (25 nM) as well as the negative controls BSA and the purified extract from the parental line ( Δxt/ft ) were added to immobilized complement proteins in microtiter plates and detected by anti-FH polyclonal antibodies. Data represent mean values ± SD from 3 independent experiments. c MFHR13, MFHR1, and FHR1 bind to the terminal complement proteins involved in MAC formation. Regulators (25 or 50 nM) were added to immobilized terminal complement proteins and bound proteins were detected by anti-His-tag antibodies. Data represent mean values ± SD from 3 or 4 independent experiments. Absorbance values from one representative experiment from ( b ) and ( c ) are shown in Supplementary Fig. . d Binding of NT-647 RED-NHS-labeled MFHR13 to C3d, C5, C7, and C9 was evaluated by Microscale Thermophoresis (MST) in fluid phase. Normalized fluorescence (ΔFnorm) was plotted against the concentration of single complement components. e MFHR13 and FHR1 inhibit the formation of the MAC on sheep erythrocytes. The inhibition by MFHR13 and FHR1 was significantly better than hFH ( P = 0.0018), eculizumab ( P < 0.0001), BSA ( P = 0.0012), or Δxt/ft . Hemolysis of sheep erythrocytes was induced with addition of C5b6, C7, C8, and C9 and release of hemoglobin was determined by measuring the absorbance at 405 nm. The average absorbance values without C5b6 and without C9 were subtracted from the positive control and samples. Lysis induced by TCC in absence of regulators was set to 100%. Data represent mean values ± SD from three independent experiments (one-way ANOVA, Bonferroni post hoc test). f MFHR13 (700 nM) inhibits MAC formation on C3b-opsonized sheep erythrocytes significantly better than FHR1 ( P = 0.0415), hFH ( P = 0.0018), eculizumab ( P < 0.0001), BSA ( P = 0.0012), cytochrome c ( P = 0.0007), and Δxt/ft . Hemolysis of C3b-coated sheep erythrocytes was induced with a mix of C5-C9 and determined by absorbance of released hemoglobin at 405 nm. Lysis without regulators was set to 100%. The average absorbance values without C5 were subtracted from all samples. Data represent mean values ± SD from three independent experiments (one-way ANOVA, Bonferroni post hoc test). **** P ≤ 0.0001, *** P ≤ 0.001, ** P ≤ 0.01, and * P ≤ 0.05, ns (no significant difference).

Article Snippet: The Bb fragments that remain bound to C3b were detected by an anti-factor B polyclonal antibody (Merck, Darmstadt, Germany), followed by HRP-conjugated rabbit anti-goat (Dako, Hamburg, Germany).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Comparison, Purification, Labeling, Microscale Thermophoresis, Fluorescence, Concentration Assay, Inhibition, Positive Control, Lysis

Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: Complement Proteins’ Western Blotting Information.

Article Snippet: 6 , CFB , 85 kDa , Goat Anti-factor B Polyclonal antibody # AF2739 (RD Systems) , 1:1000 , Human , Dnk pAb to Goat IgG (HRP) ab97120 (ABCAM) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Western Blot