Journal: Glycobiology

Article Title: Preformed mincle dimers stabilized by an interchain disulfide bond in the neck region

doi: 10.1093/glycob/cwae083

Figure Lengend Snippet: Preparation of disulfide-bonded mincle extracellular domain dimers. A) SDS-polyacrylamide gel showing test cleavage of dimerization domain from mincle extracellular domain. B) SDS-polyacrylamide gel of elution fractions from a 1-mL trehalose-Sepharose affinity column after furin cleavage. Mincle was eluted with EDTA in 0.5-mL fractions, aliquots of fractions were run on the gel in the absence of 2-mercaptoethanol, and gel was stained with Coomassie blue. C) Superdex S75 gel filtration fractionation of furin-cleaved mincle (D) SDS-polyacrylamide gel of fractions from Superdex S75 column. All gels were run in the absence of reducing agent.

Article Snippet: For test digestions, aliquots of approximately 2.5 μg of mincle in 25 μL were digested with 1–4 units of furin (New England Biolabs) at 37 °C.

Techniques: Affinity Column, Staining, Filtration, Fractionation

Journal: The Journal of Biological Chemistry

Article Title: Contradictory Functions (Activation/Termination) of Neutrophil Proteinase 3 Enzyme (PR3) in Interleukin-33 Biological Activity *

doi: 10.1074/jbc.M111.295055

Figure Lengend Snippet: Isolation of an IL-33 interacting molecule by using ligand affinity column. A, precursor IL-33 affinity chromatography column isolated urinary IL-33-interacting proteins and was visualized by silver-stained 10% SDS-PAGE. Four fractions were eluted from the IL-33 ligand affinity column. Molecular mass is indicated on the left. The arrows indicate the IL-33-binding proteins, 56-, 28-, and minor 36-kDa bands. B, mouse anti-human PR3 antibody was used for verifying urinary PR3 in the fractions from the IL-33 ligand affinity column. The molecular sizes of ∼56 (dimer form) and 28 kDa (monomer form), including the minor 36-kDa (precursor PR3) bands, were detected in the fractions. The data represent one of three independent experiments.

Article Snippet: For PR3 cleavage test with different IL-1 family ligands, commercial PR3 (100 ng from Athens Research & Technology) and the recombinant proteins of precursor human IL-33, mouse IL-33, and human IL-18 (500 ng) were incubated in 20 μl of a reaction buffer (50 m m Tris, pH 8) at room temperature.

Techniques: Isolation, Affinity Column, Staining, SDS Page, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Contradictory Functions (Activation/Termination) of Neutrophil Proteinase 3 Enzyme (PR3) in Interleukin-33 Biological Activity *

doi: 10.1074/jbc.M111.295055

Figure Lengend Snippet: PR3 enhancing precursor IL-33-induced cytokine production. A, PR3-preincubated precursor IL-33 induced IL-8 in a dose-dependent manner. PR3 (25 ng/ml) was preincubated for 5 min with various concentrations of precursor IL-33 as indicated on the bottom and then used for stimulating HMC-1 cells. B, precursor IL-33 (100 ng/ml) activity was gradually decreased along with increasing PR3 incubation times. The data represent one of three independent experiments. cont, control.

Article Snippet: For PR3 cleavage test with different IL-1 family ligands, commercial PR3 (100 ng from Athens Research & Technology) and the recombinant proteins of precursor human IL-33, mouse IL-33, and human IL-18 (500 ng) were incubated in 20 μl of a reaction buffer (50 m m Tris, pH 8) at room temperature.

Techniques: Activity Assay, Incubation, Control

Journal: The Journal of Biological Chemistry

Article Title: Contradictory Functions (Activation/Termination) of Neutrophil Proteinase 3 Enzyme (PR3) in Interleukin-33 Biological Activity *

doi: 10.1074/jbc.M111.295055

Figure Lengend Snippet: Silver staining of caspase-1 and PR3-cleaved precursor IL-33. A, process of precursor human IL-33 (250 ng/lane), mouse IL-33 (100 ng/lane), and IL-18 (500 ng/lane) was examined with caspase-1 (Casp-1) (10 units/lane). The preincubation of caspase-1 specifically cleaved precursor IL-18 and produced a single band of mature IL-18, molecular mass of 18 kDa, but both human and mouse precursor IL-33 were not affected. B, same amount of recombinant IL-1 family ligands was used for testing if these ligands were processed by PR3 (100 ng/lane). Unlike caspase-1, PR3 cleaved precursor IL-18 and produced multibands in the 2nd lane from right. The pattern of PR3-cleaved precursor IL-33 was very similar in both human and mouse. The data represent one of three independent experiments.

Article Snippet: For PR3 cleavage test with different IL-1 family ligands, commercial PR3 (100 ng from Athens Research & Technology) and the recombinant proteins of precursor human IL-33, mouse IL-33, and human IL-18 (500 ng) were incubated in 20 μl of a reaction buffer (50 m m Tris, pH 8) at room temperature.

Techniques: Silver Staining, Produced, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: Contradictory Functions (Activation/Termination) of Neutrophil Proteinase 3 Enzyme (PR3) in Interleukin-33 Biological Activity *

doi: 10.1074/jbc.M111.295055

Figure Lengend Snippet: Time-dependent reduction of a mature size of IL-33. Time course study of human precursor (Pro) IL-33 (100 ng/lane) cleavage was performed in the presence of PR3 (20 ng/lane). The precursor IL-33 was promptly reduced after 5 min of PR3 incubation. Western blot revealed that a mature (Mat) size of IL-33 was decreased by a time-dependent manner. The data represent one of three independent experiments.

Article Snippet: For PR3 cleavage test with different IL-1 family ligands, commercial PR3 (100 ng from Athens Research & Technology) and the recombinant proteins of precursor human IL-33, mouse IL-33, and human IL-18 (500 ng) were incubated in 20 μl of a reaction buffer (50 m m Tris, pH 8) at room temperature.

Techniques: Incubation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Contradictory Functions (Activation/Termination) of Neutrophil Proteinase 3 Enzyme (PR3) in Interleukin-33 Biological Activity *

doi: 10.1074/jbc.M111.295055

Figure Lengend Snippet: Alignment of human and mouse IL-33, prediction of PR3 cleavage sites, and expression of six rIL-33 proteins. A, amino acid sequence of human (Met1–Thr270) and mouse (Met1–Ile266) was aligned to predict PR3 cleavage sites by consensus sequence. The previously reported caspase cleavage sites are indicated in blue. The predicted PR3 cleavage sites are marked in red. One cleavage site is at the N terminus, and two cleavage sites are at the C terminus. Mature IL-33 from R&D Systems is indicated in green. B, six different rIL-33 proteins were expressed in E. coli as indicated at the top. The human and mouse IL-33 proteins were purified by a Talon and HPLC and then subjected to 10% SDS-PAGE. The purity of each recombinant protein was visualized by silver staining. The data represent one of three independent experiments.

Article Snippet: For PR3 cleavage test with different IL-1 family ligands, commercial PR3 (100 ng from Athens Research & Technology) and the recombinant proteins of precursor human IL-33, mouse IL-33, and human IL-18 (500 ng) were incubated in 20 μl of a reaction buffer (50 m m Tris, pH 8) at room temperature.

Techniques: Expressing, Sequencing, Purification, SDS Page, Recombinant, Silver Staining

Journal: The Journal of Biological Chemistry

Article Title: Contradictory Functions (Activation/Termination) of Neutrophil Proteinase 3 Enzyme (PR3) in Interleukin-33 Biological Activity *

doi: 10.1074/jbc.M111.295055

Figure Lengend Snippet: Biological activities of precursor IL-33 and three new rIL-33 proteins from PR3-cleaved forms. A, biological activities of human IL-33 (20 ng/ml) proteins from three recombinant PR3-cleaved forms, including pro-IL-33 were examined with human HMC-1 (A) and Raw 264.7 (B) cells. Recombinant IL-33/p1 induced cytokines, but the recombinant protein of precursor IL-33, IL-33/p2, and IL-33/p3 were not active. The data represent one of three independent experiments. cont, control.

Article Snippet: For PR3 cleavage test with different IL-1 family ligands, commercial PR3 (100 ng from Athens Research & Technology) and the recombinant proteins of precursor human IL-33, mouse IL-33, and human IL-18 (500 ng) were incubated in 20 μl of a reaction buffer (50 m m Tris, pH 8) at room temperature.

Techniques: Recombinant, Control