Journal: medRxiv

Article Title: Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation

doi: 10.1101/2020.03.29.20041962

Figure Lengend Snippet: The N proteins of SARS-CoV, MERS-CoV and SARS-CoV-2 bind to MASP-2. (A) Lysates of 293T cells transfected with indicated plasmids were subjected to anti-Flag immunoprecipitation in the presence of 2 mM CaCl 2 or 1 mM EDTA, and analyzed by immunoblotting. IgG immnuoprecipitates were used as a negative control. (B) Lysates of 293T cells transfected with indicated plasmids were mixed with human serum (HS) and mouse serum (MS) and subjected to immunoprecipitation with anti-Flag agarose beads. The absorbates were probed with indicated antibodies. Purified recombinant MASP-2 was loaded as a marker. (C)-(E) Lysates of 293T cells transfected with indicated plasmids were subjected to anti-Flag immunoprecipitation in the presence of 2 mM CaCl 2 and analyzed by immunoblotting.

Article Snippet: Immunoblot analysis was performed with horseradish peroxidase (HRP)-conjugated anti-Flag (Sigma), anti-β-actin (Sigma), anti-green fluorescent protein (GFP) (Clontech), anti-MASP-2 (Santa Cruz), anti-C4α (Santa Cruz), HRP-conjugated anti-Myc (Santa Cruz), and goat anti-mouse immunoglobulin G (IgG) (Amersham/Pharmacia) antibodies.

Techniques: Transfection, Immunoprecipitation, Western Blot, Negative Control, Purification, Recombinant, Marker

Journal: medRxiv

Article Title: Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation

doi: 10.1101/2020.03.29.20041962

Figure Lengend Snippet: The N proteins induce MASP-2 auto-activation and C4 cleavage. (A) Lysates from cells expressing indicated proteins were mixed with purified N or MBL and subjected to anti-Flag immunoprecipitation and analyzed by immunoblotting. (B) Purified MASP-2-Flag was incubated with/without N, MBL, and mannan at 37°C for 12 hr. Cleaved MASP-2 was probed with an anti-Flag antibody. (C) Purified MASP-2 and N proteins with/without anti-N monoclonal antibody were incubated with pre-conjugated MBL in mannan-coated plates at 4°C. Binding of MASP-2 was detected with an anti-MASP-2 antibody. (D) C4 was incubated with indicated components at 37°C for 0, 2, 6, 12 and 24 hr. C4 and a cleaved C4alpha’ fragment were detected with anti-C4alpha chain antibody. (E) C4 was incubated with indicated components at 37°C for 1 hr. C4 cleavage was detected as mentioned above. Ab, anti-MASP-2 antibody. (F)-(H) C4b deposition in relation to the concentration of N proteins of SARS-CoV, HCoV-229E, SARS-CoV-2, and MERS-CoV.

Article Snippet: Immunoblot analysis was performed with horseradish peroxidase (HRP)-conjugated anti-Flag (Sigma), anti-β-actin (Sigma), anti-green fluorescent protein (GFP) (Clontech), anti-MASP-2 (Santa Cruz), anti-C4α (Santa Cruz), HRP-conjugated anti-Myc (Santa Cruz), and goat anti-mouse immunoglobulin G (IgG) (Amersham/Pharmacia) antibodies.

Techniques: Activation Assay, Expressing, Purification, Immunoprecipitation, Western Blot, Incubation, Binding Assay, Concentration Assay

Journal: medRxiv

Article Title: Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation

doi: 10.1101/2020.03.29.20041962

Figure Lengend Snippet: N proteins potentiate LPS-induced pneumonia in mice. (A) BALB/c mice (10 mice/group) were infected with 1×10 9 PFU Ad-SARS N/Ad-null or a saline control, and LPS (5 mg/kg) was given on the 6th days post infection (d.p.i.). Antibodies (200 μg/kg) or C1INH (4 mg/kg) was injected 30 min before LPS injection. The fatality of mice was noted. (B) Lung paraffin sections were analyzed by HE staining. (C) Mice were infected with 1×10 8 PFU Ad-SARS N/Ad-null, and LPS (5 mg/kg) was given by on 6 d.p.i. Mice were sacrificed after LPS challenged for 6 hr. Frozen lung sections were stained with anti-C4b antibody. (D) SARS-CoV N and MASP-2 complex formation in frozen lung sections was measured by in situ PLA, as indicated by the red signals. Deposited C3 fragments were stained with FITC-labeled anti-C3c antibodies (green). Scale bar, 50 μm. (E) Mice were pre-infected with 1×10 9 PFU Ad-MERS N/Ad-null and treated with LPS, anti-MASP-2 antibody or C1INH as mentioned above. The mice survival curve was plotted. (F) Masp2 -/- and Masp2 +/+ C57BL/6N mice were infected N-expressing adenovirus and injected with LPS as mentioned above. The mice survival curve was plotted.

Article Snippet: Immunoblot analysis was performed with horseradish peroxidase (HRP)-conjugated anti-Flag (Sigma), anti-β-actin (Sigma), anti-green fluorescent protein (GFP) (Clontech), anti-MASP-2 (Santa Cruz), anti-C4α (Santa Cruz), HRP-conjugated anti-Myc (Santa Cruz), and goat anti-mouse immunoglobulin G (IgG) (Amersham/Pharmacia) antibodies.

Techniques: Infection, Injection, Staining, In Situ, Labeling, Expressing

Journal: medRxiv

Article Title: Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation

doi: 10.1101/2020.03.29.20041962

Figure Lengend Snippet: Schematic representation of MBL pathway over-activated by N protein of SARS/ MERS-CoV or SARS-CoV-2. (A) Virus binds to cell surface and S protein actives MBL. (B) Virus enters cells and expresses viral proteins including N protein. (C) N proteins release by secretion or after cells and virus lysis. (D) The extracellular soluble N protein dimmers interact with MASP-2, induce MASP-2 auto-activation and binding to MBL. (E) The accelerated activation of MASP-2 induces downstream complements cascades over-activation of the MBL pathway, and promotes cell lysis and N protein releases by secretion or complement mediated cytotoxicity, which may result in uncontrolled tissue damage and inflammation.

Article Snippet: Immunoblot analysis was performed with horseradish peroxidase (HRP)-conjugated anti-Flag (Sigma), anti-β-actin (Sigma), anti-green fluorescent protein (GFP) (Clontech), anti-MASP-2 (Santa Cruz), anti-C4α (Santa Cruz), HRP-conjugated anti-Myc (Santa Cruz), and goat anti-mouse immunoglobulin G (IgG) (Amersham/Pharmacia) antibodies.

Techniques: Lysis, Activation Assay, Binding Assay, Flow Cytometry Complement-Mediated Cytotoxicity Assay

Journal: Molecular Biology Reports

Article Title: Association between mannose binding lectin gene polymorphisms and clinical severity of COVID-19 in children

doi: 10.1007/s11033-023-08524-z

Figure Lengend Snippet: Serum MBL and MASP-2 levels according to the clinical features of the patients

Article Snippet: The human MBL and MASP-2 ELISA kits measured serum MBL and MASP-2 levels in samples from patients (BT LAB, China).

Techniques:

Journal: Molecular Biology Reports

Article Title: Association between mannose binding lectin gene polymorphisms and clinical severity of COVID-19 in children

doi: 10.1007/s11033-023-08524-z

Figure Lengend Snippet: Serum MBL and MASP-2 levels, according to the Codon 54 polymorphisms

Article Snippet: The human MBL and MASP-2 ELISA kits measured serum MBL and MASP-2 levels in samples from patients (BT LAB, China).

Techniques: