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: Cell reports

Article Title: Human IL-10-producing B cells have diverse states that are induced from multiple B cell subsets

doi: 10.1016/j.celrep.2022.110728

Figure Lengend Snippet:

Article Snippet: anti-IgA (polyclonal) - 148 Nd , Fluidigm , Cat # 3148007B.

Techniques: Purification, Recombinant, Formulation, Blocking Assay, Antibody Labeling, Software