Journal: The Journal of Experimental Medicine

Article Title: Potentiation of C1 Esterase Inhibitor by StcE, a Metalloprotease Secreted by Escherichia coli O157:H7

doi: 10.1084/jem.20030255

Figure Lengend Snippet: C1-INH binds erythrocyte surfaces in the presence of StcE. (A) 8 μg C1-INH was untreated or treated with 1 μg StcE′-His overnight at room temperature. C5-deficient human serum was added to opsonized sheep erythrocytes in the presence of 8 μg C1-INH, 1 μg StcE′-His, C1-INH, and StcE′-His, or buffer alone (mock) for 10 min at 37°C. Erythrocytes were washed and polyclonal goat anti–human C1-INH IgG was added to cells. C1-INH binding was detected with FITC-conjugated anti–goat IgG in a flow cytometer. (B) 8 μg C1-INH was incubated with or without 1 μg StcE′-His overnight at room temperature before the addition of 50 mM imidazole and Ni-NTA agarose beads to remove StcE′-His from the sample. Opsonized sheep erythrocytes were added for 10 min at 37°C and C1-INH binding was detected as described. As a control for StcE-treated C1-INH binding, StcE′-His was not removed from one sample before analysis. (C) Increasing concentrations of C1-INH (0.25–16 μg) were mixed with 1 μg StcE′ E435D-His overnight at room temperature before the addition of opsonized erythrocytes. C1-INH binding was detected in a flow cytometer as described above, and the geometric mean fluorescence for each sample was plotted against the corresponding concentration of C1-INH. (D) 5 μg C1-INH was untreated or treated with 5 μg StcE′-His or StcE′ E435D-His for 10 min before being immunoprecipitated with an anti–C1-INH Ab. Precipitated proteins were separated by reducing SDS-PAGE, transferred to nitrocellulose, and probed with an anti-StcE Ab as described in Materials and Methods.

Article Snippet: Cells were washed with VBS 2+ and incubated on ice for 30 min with polyclonal goat anti–human IgG against C1-INH (Cedarlane Laboratories).

Techniques: Binding Assay, Flow Cytometry, Incubation, Control, Fluorescence, Concentration Assay, Immunoprecipitation, SDS Page

Journal: The Journal of Experimental Medicine

Article Title: Potentiation of C1 Esterase Inhibitor by StcE, a Metalloprotease Secreted by Escherichia coli O157:H7

doi: 10.1084/jem.20030255

Figure Lengend Snippet: Cleavage of C1-INH by StcE is not necessary to bind erythrocytes or provide protection against classical complement. (A) Classical complement-mediated erythrocyte lysis was determined as described in Materials and Methods in the presence of 8 μg C1-INH, C1-INH and 1 μg StcE′-His, or C1-INH and an enzymatic point mutant of StcE, 1 μg StcE′ E435D-His (*, P < 0.005; unpaired t test). (B) 2 μg C1-INH was untreated or treated with 1 μg StcE′-His or StcE′ E435D-His before the addition of sheep erythrocytes as described in Materials and Methods. Erythrocytes were washed and polyclonal goat anti–human C1-INH IgG was added to cells. C1-INH binding was detected with FITC-conjugated anti–goat IgG in a flow cytometer.

Article Snippet: Cells were washed with VBS 2+ and incubated on ice for 30 min with polyclonal goat anti–human IgG against C1-INH (Cedarlane Laboratories).

Techniques: Lysis, Mutagenesis, Binding Assay, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Potentiation of C1 Esterase Inhibitor by StcE, a Metalloprotease Secreted by Escherichia coli O157:H7

doi: 10.1084/jem.20030255

Figure Lengend Snippet: StcE interacts with the aminoterminal domain of C1-INH. (A) StcE-treated C1-INH is nonreactive with mAbs against the NH 2 terminus of and RCL-inserted C1-INH. 1 μg virgin C1-INH was untreated or treated with 1 μg StcE′-His or 2 μg kallikrein overnight at room temperature, separated by electrophoresis on 8% reducing SDS-PAGE gels, transferred to nitrocellulose, and analyzed with a polyclonal anti–human C1-INH Ab (left), mAb 3C7 (middle), or mAb 4C3 (right). (B) StcE does not cleave C-serp(98), a recombinant C1-INH molecule truncated at amino acid 98. COS-7 cells were transfected with hC1-INH/pcDNA3.1(−) or C-serp(98)/pcDNA3.1(−) and metabolically labeled with [ 35 S]methionine. 100 μl supernatants were untreated or treated with 10 μg StcE′-His overnight, immunoprecipitated with polyclonal anti–human C1-INH IgG protein A–Sepharose, and separated by electrophoresis on a 10% reducing SDS-PAGE gel.

Article Snippet: Cells were washed with VBS 2+ and incubated on ice for 30 min with polyclonal goat anti–human IgG against C1-INH (Cedarlane Laboratories).

Techniques: Electrophoresis, SDS Page, Recombinant, Transfection, Metabolic Labelling, Labeling, Immunoprecipitation

Journal: iScience

Article Title: Racial heterogeneity of IgA1 hinge-region O -glycoforms in patients with IgA nephropathy

doi: 10.1016/j.isci.2022.105223

Figure Lengend Snippet: Comparison of serum Gd-IgA1 levels among four groups: J-HC, J-IgAN, G-HC, and G-IgAN The medians (quartile range) of Gd-IgA1 levels were 0.40 (0.23–0.52), 0.86 (0.55–1.25), 0.73 (0.38–1.17), and 1.29 (0.85–1.96), respectively. Gd-IgA1 levels differed significantly among the four groups (Kruskal-Wallis test, p < 0.001). Gd-IgA1 levels were significantly higher in J-IgAN, G-HC, and G-IgAN than in the reference group (J-HC) (Dunn’s correction, p < 0.001, p = 0.005, and p < 0.001, respectively). Gd-IgA1, galactose-deficient IgA1; J-HC, Japanese healthy control; J-IgAN, Japanese patients with IgAN; G-HC, Greek healthy control; G-IgAN, Greek patients with IgAN; ∗∗, 0.001 ≤ p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: IgA1 was purified from 100 μL serum of patients with IgAN and HCs using affinity chromatography with anti-human IgA (0855068, MP Biomedicals, Irvine, CA, USA) coupled to a HiTrap NHS-activated HP column (17071601, GE Healthcare, Chicago, IL, USA).

Techniques:

Journal: iScience

Article Title: Racial heterogeneity of IgA1 hinge-region O -glycoforms in patients with IgA nephropathy

doi: 10.1016/j.isci.2022.105223

Figure Lengend Snippet: Comparison of desialylated IgA1 HR O -glycoforms between healthy controls (HCs) and patients with IgA nephropathy (IgAN) in Japanese and Greek cohorts (A) IgA1 HR O -glycoforms of the Japanese cohort. (B) IgA1 HR O -glycoforms of the Greek cohort. The medians of relative abundance (%) in each O -glycoform are represented by black bars. Relative abundance of IgA1 HR with 3GalNAc3Gal increased significantly, whereas that of 5GalNAc4Gal decreased significantly in J-IgAN compared with that in J-HC (Mann-Whitney test, p < 0.001 and Student’s t test, p = 0.040, respectively). In the Greek cohort, the relative abundance of IgA1 HR with 3GalNAc2Gal and 5GalNAc3Gal was significantly higher (Student’s t test, p = 0.008 and Mann-Whitney test, p = 0.043, respectively) and that of 6GalNAc4Gal and 6GalNAc5Gal was significantly lower in G-IgAN than in G-HC (Student’s t test, p = 0.006 and = 0.001, respectively). J-HC, Japanese-HCs; J-IgAN, Japanese patients with IgAN; G-HC, Greek HCs; G-IgAN, Greek patients with IgAN; ∗, 0.01 ≤ p < 0.05; ∗∗, 0.001 ≤ p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: IgA1 was purified from 100 μL serum of patients with IgAN and HCs using affinity chromatography with anti-human IgA (0855068, MP Biomedicals, Irvine, CA, USA) coupled to a HiTrap NHS-activated HP column (17071601, GE Healthcare, Chicago, IL, USA).

Techniques: MANN-WHITNEY

Journal: iScience

Article Title: Racial heterogeneity of IgA1 hinge-region O -glycoforms in patients with IgA nephropathy

doi: 10.1016/j.isci.2022.105223

Figure Lengend Snippet: Representative mass spectra of the desialylated tryptic fragments of IgA1 HR O -glycoforms acquired from Japanese (A) and Greek (B) patients with IgAN The monoisotopic m/z value of the HR O -glycopeptide ions and the number of sugar moieties assigned are shown above the individual peaks. The HR O -glycoforms, the levels of which were higher in Japanese patients than in Greek patients, are represented by upward arrows above the individual peaks in the mass spectra of Japanese patients. The HR O -glycoforms, the levels of which were elevated in Greek patients than in Japanese patients, are represented by upward arrows in the mass spectra of the Greek patients. Comparison of two groups was performed using Student’s t test or Mann-Whitney test depending on whether the variables were distributed normally. ∗, 0.01

Article Snippet: IgA1 was purified from 100 μL serum of patients with IgAN and HCs using affinity chromatography with anti-human IgA (0855068, MP Biomedicals, Irvine, CA, USA) coupled to a HiTrap NHS-activated HP column (17071601, GE Healthcare, Chicago, IL, USA).

Techniques: MANN-WHITNEY

Journal: iScience

Article Title: Racial heterogeneity of IgA1 hinge-region O -glycoforms in patients with IgA nephropathy

doi: 10.1016/j.isci.2022.105223

Figure Lengend Snippet: IgA1 glycoforms expressed based on a specific monosaccharide per HR (A) Relative abundance of IgA1 HR peptide with 3 GalNAc residues. This HR glycoform was higher in J-IgAN and G-IgAN than in the reference group (J-HC) based on Dunn’s multiple comparison test (p = 0.008 and p = 0.001, respectively). (B) Relative abundance of IgA1 HR peptide with 4 GalNAc residues. This HR glycoform was higher in G-IgAN than in J-HC (Dunnett’s correction p = 0.017). (C) Relative abundance of IgA1 HR peptide with 5 GalNAc residues. (D) Relative abundance of IgA1 HR peptide with 6 GalNAc residues. This HR glycoform was lower in G-IgAN than in J-HC (Dunnett’s correction p < 0.001). (E) Mean number of GalNAc per HR. The levels were lower in G-IgAN than in J-HC (Dunn’s correction p = 0.003. (F) Mean number of Gal per HR. (G) Mean number of Gd-glycan per HR. The data are shown in the scatter dot plot (with line drawn at the median). GalNAc, N -acetylgalactosamine; Gal, galactose; Gd-glycan, galactose-deficient-glycan; HR, hinge region; J-HC, Japanese healthy controls; J-IgAN, Japanese patients with IgAN; G-HC, Greek healthy controls; G-IgAN, Greek patients with IgAN. ∗, 0.01 ≤ p < 0.05; ∗∗, 0.001 ≤ p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: IgA1 was purified from 100 μL serum of patients with IgAN and HCs using affinity chromatography with anti-human IgA (0855068, MP Biomedicals, Irvine, CA, USA) coupled to a HiTrap NHS-activated HP column (17071601, GE Healthcare, Chicago, IL, USA).

Techniques:

Journal: iScience

Article Title: Racial heterogeneity of IgA1 hinge-region O -glycoforms in patients with IgA nephropathy

doi: 10.1016/j.isci.2022.105223

Figure Lengend Snippet:

Article Snippet: IgA1 was purified from 100 μL serum of patients with IgAN and HCs using affinity chromatography with anti-human IgA (0855068, MP Biomedicals, Irvine, CA, USA) coupled to a HiTrap NHS-activated HP column (17071601, GE Healthcare, Chicago, IL, USA).

Techniques: Software

Journal: Cell

Article Title: Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection

doi: 10.1016/j.cell.2021.01.035

Figure Lengend Snippet:

Article Snippet: Five-fold serial dilutions of polyclonal macaque (Molecular Innovations) or human IgG, starting at a concentration of 1 μg/mL, were added to wells containing the coated goat anti-Human IgG λ and κ.

Techniques: Purification, Recombinant, Mass Spectrometry, Sequencing, Ligation, Luciferase, Enzyme-linked Immunospot, Plasmid Preparation, Software, Chromatography, Luminex, Expressing