Journal: PLoS ONE

Article Title: Hepatitis C Virus NS3/4A Protease Inhibits Complement Activation by Cleaving Complement Component 4

doi: 10.1371/journal.pone.0082094

Figure Lengend Snippet: (A) HCV NS3/4A protease, core, or NS5 was added to C4, and the products were separated by SDS-PAGE and subjected to CBB staining. Two approximately 17-kDa proteins (Fragment F1 and F2) and a 15-kDa protein (Fragment F3) were detected after incubation of C4 with HCV NS3/4A protease, but not after incubation with core or NS5. (B) Amino acid sequence of aa 1451-1620 region of C4. Protein fragments were analyzed by N-terminal peptide sequencing. The sequences of the N-termini of the 17-kDa proteins (Fragment F1 and F2) were SAEVCQCA and AEGKCPRQ, which are located at aa 1584–1591 and 1591–1598 in C4, respectively. The sequence of the N-terminus of the 15-kDa protein (Fragment F3) was EAPKVVEE, which is located at aa 1454–1461 in C4. (C) Schematic representation of C4γ chain, and Fragment F1, F2 and F3.

Article Snippet: Goat anti-human complement C4 antibody (MP Biomedicals, Santa Ana, CA) was bound to protein G–agarose beads (Thermo Scientific, Rockford, IL) in binding buffer (0.5% Nonidet P-40, 25 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA and protease inhibitor cocktail [Roche, Basel, Switzerland]) for 1 h at room temperature.

Techniques: SDS Page, Staining, Incubation, Sequencing

Journal: PLoS ONE

Article Title: Hepatitis C Virus NS3/4A Protease Inhibits Complement Activation by Cleaving Complement Component 4

doi: 10.1371/journal.pone.0082094

Figure Lengend Snippet: C4 was incubated in the presence or absence of HCV NS3/4A protease, and then C1-sensitized EA (EAC1) was added (EAC1-C4). After washing, C2 was added to form EAC1-C4-C2, and the complex was resuspended in C4d-GPS. The absorbance of the centrifuged supernatant was determined at 415 nm. The grade of hemolysis decreased in the presence of NS3/4A protease in a dose-dependent manner. All measurements were performed in triplicate, and data are expressed as means ± SD.

Article Snippet: Goat anti-human complement C4 antibody (MP Biomedicals, Santa Ana, CA) was bound to protein G–agarose beads (Thermo Scientific, Rockford, IL) in binding buffer (0.5% Nonidet P-40, 25 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA and protease inhibitor cocktail [Roche, Basel, Switzerland]) for 1 h at room temperature.

Techniques: Incubation

Journal: PLoS ONE

Article Title: Hepatitis C Virus NS3/4A Protease Inhibits Complement Activation by Cleaving Complement Component 4

doi: 10.1371/journal.pone.0082094

Figure Lengend Snippet: (A) VX950 was added to HCV NS3/4A protease at the indicated concentrations, and then C4 was added. Proteins were separated by SDS-PAGE for CBB staining. The three C4-derived fragments of 17 kDa and 15 kDa produced by NS3/4A protease action could not be detected after pretreatment with VX950, and this change was accompanied by an increased concentration of the 32-kDa C4γ chain. (B) The C4γ, 17-kDa, and 15-kDa bands were quantified by densitometric analysis using the Image J software. (C) C4 was incubated in the presence or absence of HCV NS3/4A or VX950, and then C1-sensitized EA (EAC1) was added (EAC1–C4). C2 and C4d-GPS were then added, and the absorbance of the supernatant was determined at 415 nm. Hemolysis was inhibited by NS3/4A protease and this inhibition was blocked by VX950. All measurements were made in triplicate, and data are expressed as means ± SD.

Article Snippet: Goat anti-human complement C4 antibody (MP Biomedicals, Santa Ana, CA) was bound to protein G–agarose beads (Thermo Scientific, Rockford, IL) in binding buffer (0.5% Nonidet P-40, 25 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA and protease inhibitor cocktail [Roche, Basel, Switzerland]) for 1 h at room temperature.

Techniques: SDS Page, Staining, Derivative Assay, Produced, Concentration Assay, Software, Incubation, Inhibition

Journal: PLoS ONE

Article Title: Hepatitis C Virus NS3/4A Protease Inhibits Complement Activation by Cleaving Complement Component 4

doi: 10.1371/journal.pone.0082094

Figure Lengend Snippet: (A) 293T cells were transfected with the indicated plasmids. Anti-C4 immunoprecipitates (IP) of supernatants were separated by SDS-PAGE and analyzed by immunoblotting with anti-C4γ antibody. Detergent-soluble cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with anti-HA and anti-GAPDH antibodies. (B) 293T cells were transfected with the indicated plasmids. Culture supernatants were analyzed by immunoblotting with anti-C4γ antibody. Anti-C4 immunoprecipitates (IP) of supernatants were analyzed by immunoblotting with anti-C4 antibody. Detergent-soluble cell lysates were analyzed by immunoblotting with anti-HA and anti-GAPDH antibodies. (C) Huh7.5.1 cells were mock-infected or infected with HCVcc at a multiplicity of infection of 2 for 6 h, followed by mock-transfection or transfection with C4 expression plasmid. Culture supernatants and cell lysates were analyzed as described in (A) and (B). The anti-C4γ antibody was not appropriate for immunoblotting of IP samples derived from Huh7.5.1 cultures because of unavoidable nonspecific cross-reaction. * indicates non-specific reactions in (A) – (C).

Article Snippet: Goat anti-human complement C4 antibody (MP Biomedicals, Santa Ana, CA) was bound to protein G–agarose beads (Thermo Scientific, Rockford, IL) in binding buffer (0.5% Nonidet P-40, 25 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA and protease inhibitor cocktail [Roche, Basel, Switzerland]) for 1 h at room temperature.

Techniques: Transfection, SDS Page, Western Blot, Infection, Expressing, Plasmid Preparation, Derivative Assay

Journal: Cell reports

Article Title: Complement C4A Regulates Autoreactive B Cells in Murine Lupus

doi: 10.1016/j.celrep.2020.108330

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Goat IgG fraction to mouse complement C3 , MP Biomedicals , Cat# ICN55463, RRID:AB_2334481.

Techniques: Purification, Recombinant, Software

Journal: Med (New York, N.y.)

Article Title: Reduced blood-stage malaria growth and immune correlates in humans following RH5 vaccination

doi: 10.1016/j.medj.2021.03.014

Figure Lengend Snippet: Antibody immunogenicity of RH5.1/AS01 B (A) Timing of immunizations and follow-up in groups 1–4. All antigen doses were formulated in 0.5 mL AS01 B . (B and C) Median and individual anti-RH5_FL serum total IgG responses 14 days after two vaccinations (Vacs; day 42, B) and after three Vacs (day 70 or day 196, C). Both datasets were analyzed separately by Kruskal-Wallis test with Dunn’s multiple comparisons test; ∗∗p < 0.01. Historical data for the VV-RH5 vaccine were not included in the analysis and are shown for comparison only. (D) In vitro GIA of purified IgG assessed at 10 mg/mL against 3D7 clone P. falciparum parasites. Individual data and medians are shown for each group at the stated time-point; pooled sera were used for each group at baseline (day 0). Historical data for VV-RH5 were included as before. (E) Dilution series of purified IgG for all group 1–4 samples starting from 10 mg/mL. (F) Relationship between GIA data from the dilution series shown in (E) and concentration of anti-RH5_FL purified IgG used in the assay as measured by ELISA. A non-linear regression curve is shown for all samples combined (solid line, r 2 = 0.96, n = 279). The EC 50 (concentration of anti-RH5_FL polyclonal IgG that gives 50% GIA, dashed line) was calculated.

Article Snippet: FITC-conjugated, goat anti-guinea pig complement C3 polyclonal antibody , MP Biomedical , 0855385.

Techniques: In Vitro, Purification, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Med (New York, N.y.)

Article Title: Reduced blood-stage malaria growth and immune correlates in humans following RH5 vaccination

doi: 10.1016/j.medj.2021.03.014

Figure Lengend Snippet:

Article Snippet: FITC-conjugated, goat anti-guinea pig complement C3 polyclonal antibody , MP Biomedical , 0855385.

Techniques: Recombinant, Purification, Plasmid Preparation, Software, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Flow Cytometry

Journal: eNeuro

Article Title: Noncanonical Activity of Tissue Inhibitor of Metalloproteinases 2 (TIMP2) Improves Cognition and Synapse Density in Aging

doi: 10.1523/ENEURO.0031-23.2023

Figure Lengend Snippet: Key resources

Article Snippet: The ability of the protein constructs to inhibit MMP2 was analyzed using the SensoLyte 520 MMP-2 Assay kit (AS-71151, AnaSpec) and recombinant human MMP-2 pro-enzyme (Hu-pro-MMP2.0, 902-MP-010, R&D Systems; Hu-pro-MMP2.2, AS-72005, AnaSpec).

Techniques: Recombinant, Diagnostic Assay, Plasmid Preparation, Staining, Extraction, Sterility, Saline, Construct, Electron Microscopy, Expressing, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Multiplex Assay, Transfection, Sequencing, Software, Control

Journal: eNeuro

Article Title: Noncanonical Activity of Tissue Inhibitor of Metalloproteinases 2 (TIMP2) Improves Cognition and Synapse Density in Aging

doi: 10.1523/ENEURO.0031-23.2023

Figure Lengend Snippet: The TIMP2 constructs had distinct MMP inhibitory profiles

Article Snippet: The ability of the protein constructs to inhibit MMP2 was analyzed using the SensoLyte 520 MMP-2 Assay kit (AS-71151, AnaSpec) and recombinant human MMP-2 pro-enzyme (Hu-pro-MMP2.0, 902-MP-010, R&D Systems; Hu-pro-MMP2.2, AS-72005, AnaSpec).

Techniques: Construct, Inhibition

Journal: eNeuro

Article Title: Noncanonical Activity of Tissue Inhibitor of Metalloproteinases 2 (TIMP2) Improves Cognition and Synapse Density in Aging

doi: 10.1523/ENEURO.0031-23.2023

Figure Lengend Snippet: The alanine insertion into TIMP2 prevented MMP inhibitory activity at biologically relevant concentrations

Article Snippet: The ability of the protein constructs to inhibit MMP2 was analyzed using the SensoLyte 520 MMP-2 Assay kit (AS-71151, AnaSpec) and recombinant human MMP-2 pro-enzyme (Hu-pro-MMP2.0, 902-MP-010, R&D Systems; Hu-pro-MMP2.2, AS-72005, AnaSpec).

Techniques: Activity Assay

Journal: eNeuro

Article Title: Noncanonical Activity of Tissue Inhibitor of Metalloproteinases 2 (TIMP2) Improves Cognition and Synapse Density in Aging

doi: 10.1523/ENEURO.0031-23.2023

Figure Lengend Snippet: Characterization of TIMP2-MMP binding of the TIMP2 constructs

Article Snippet: The ability of the protein constructs to inhibit MMP2 was analyzed using the SensoLyte 520 MMP-2 Assay kit (AS-71151, AnaSpec) and recombinant human MMP-2 pro-enzyme (Hu-pro-MMP2.0, 902-MP-010, R&D Systems; Hu-pro-MMP2.2, AS-72005, AnaSpec).

Techniques: Binding Assay