Journal: bioRxiv

Article Title: Neuronal pentraxin Nptx2 regulates complement activity in the brain

doi: 10.1101/2022.09.22.509106

Figure Lengend Snippet: A) Microtiter wells were coated with BSA, IgM or neuronal pentraxins (Nptx1, Nptx2, Nptxr) as indicated and incubated with C1q. Binding of C1q to the wells was detected using an anti-C1q antibody. p values were determined by one-way ANOVA. B) Microtiter wells were coated with BSA or complement proteins (C1q, C2, C3, C4) as indicated. Binding of his-tagged neuronal pentraxins was monitored using an anti-His antibody. p values were determined by Two-way ANOVA. C) Representative images of negative staining electron microscopy of purified C1q, Nptx2, and C1q-Nptx2 complex. Scale bar: 10 nm. D) C1q cell-surface binding assays with a stable tetracycline-inducible CHO cell-line that co-expresses Nptxr and Nptx2. C1q proteins were added to the fresh culture medium, and bound C1q was visualized by immunofluorescence labeling (red). Expression of Nptxr was detected with a Nptxr-specific antibody (green). Scale bar: 50 µm. E) Cell-surface binding to Nptxr/x2-expressing CHO cells with C1q as ligand. Stable inducible CHO cells expressing Nptxr/x2 were incubated with C1q at indicated concentrations. Bound C1q was detected with an anti-C1q antibody. The signal shown was subtracted from the background in CHO cells without induced Nptxr expression. p value was determined by Two-way ANOVA. F) Neuronal pentraxin inhibits hemolysis of sheep erythrocytes. Purified Nptx1, Nptx2, Nptxr or ApoE3 was incubated in normal human serum (NHS), which was activated via CCP-specific GVB++ buffer, and lysis of sheep erythrocytes was analyzed by measuring released hemoglobin at 415 nm. G) Nptxr inhibits CCP and activated C3 deposition. 1% NHS supplemented with recombinant Nptxr was added to IgM-coated microtiter plates and activated C3 deposition was measured using specific antibodies. Heat-inactivated NHS and C1q-depleated human serum were used as negative control. p values were determined by one-way ANOVA. Data are presented as mean ± SEM. Each dot represents data from individual samples.

Article Snippet: Purified human complement proteins (4 μg/mL, Complement Technology) were immobilized and were treated with His-tagged recombinant neuronal pentraxin proteins (4 μg/mL, R&D Systems).

Techniques: Incubation, Binding Assay, Negative Staining, Electron Microscopy, Purification, Immunofluorescence, Labeling, Expressing, Lysis, Recombinant, Negative Control

Journal: bioRxiv

Article Title: Neuronal pentraxin Nptx2 regulates complement activity in the brain

doi: 10.1101/2022.09.22.509106

Figure Lengend Snippet: A) Microtiter wells were coated with 2µg/ml IgM or 0.625 – 10 µg/ml Nptxr and incubated with various concentrations of C1q as indicated. Bound C1q was detected using an anti-C1q antibody. B) Purified Nptx2 pentraxin domain (X2-PD) coupled to CNBr-activated Sepharose 4B was incubated with purified C1q. Proteins were separated by SDS-PAGE and visualized by Coomassie blue staining. C) Quantification of hemolysis of sheep erythrocytes in presence of increased NHS concentrations. Lysis of sheep erythrocytes was monitored by measuring released hemoglobin at 415 nm. p values were determined by one-way ANOVA. D) Purified XR-PD was added to NHS in a CCP-specific buffer. Lysis of sheep erythrocytes was monitored by measuring released hemoglobin at 415 nm. p values were determined by two-way ANOVA. E) Survival of E. coli in presence of purified XR-PD in NHS. E. coli survival was analyzed by counting colony forming units. Heat inactivated NHS was used as a negative control. p values were determined by one-way ANOVA. Data are presented as mean ± SEM. Each dot represents data from individual samples.

Article Snippet: Purified human complement proteins (4 μg/mL, Complement Technology) were immobilized and were treated with His-tagged recombinant neuronal pentraxin proteins (4 μg/mL, R&D Systems).

Techniques: Incubation, Purification, SDS Page, Staining, Lysis, Negative Control