Journal: The Journal of biological chemistry

Article Title: DNA-mediated proteolysis by neutrophil elastase enhances binding activities of the HMGB1 protein.

doi: 10.1016/j.jbc.2022.102577

Figure Lengend Snippet: Figure 6. Potential roles of the DNA-mediated proteolytic processing of HMGB1 by neutrophil elastase in NETs. Due to the enhanced binding activities of the processed HMGB1 protein, this processing may promote (1) TLR4 signaling, (2) binding to biofilm DNA, and (3) DNA sensing by cGAS. Due to the loss of residues 177–215, the processing of HMGB1 may diminish (4) RAGE signaling and (5) nuclear localization. NET, neutrophil extracellular trap.

Article Snippet: Lyophilized TLR4 MD-2 complex was purchased from R&D Systems (catalog no.: #3146-TM-050).

Techniques: Binding Assay

Journal: ACS Nano

Article Title: Force Nanoscopy Demonstrates Stress-Activated Adhesion between Staphylococcus aureus Iron-Regulated Surface Determinant Protein B and Host Toll-like Receptor 4

doi: 10.1021/acsnano.4c12648

Figure Lengend Snippet: IsdB-TLR4 strength confirmed by anti-TLR4 blocking and S. aureus strain lacking IsdB. (a) Rupture force histograms of 2 representative Δ spa cells, cultured in RPMI medium, obtained by recording force–distance curves in PBS at a retraction speed of 1 μm/s between TLR4-functionalized AFM tips before ( n = 188 and 276 adhesive curves, for cell #1 and cell #2, respectively) and after blocking with 100 μg/mL of anti-TLR4 monoclonal antibody ( n = 98 and 65 adhesive curves, for cell #1 and cell #2, respectively). (b) Rupture force and rupture length histograms of a representative S. aureus cell expressing IsdB (WT, n = 200 adhesive curves) cultured in RPMI. (c) Force data of a representative S. aureus cell lacking IsdB (Δ isdB , n = 95 adhesive curves) cultured in RPMI. Schemes of the SMFS setups and representative retraction force profiles are shown as insets. Box plots comparing (d) binding frequency and (e) rupture forces obtained for Δ spa cells before and after blocking with anti-TLR4 monoclonal antibody, after tip treatment with monoclonal mouse IgG as a negative control, WT and Δ isdB strains ( n = 8, 8, 10, 13, and 9 cells, respectively). Means are represented by stars, medians by lines, boxes indicate the 25–75% quartiles, and whiskers the standard deviation. P -values were determined using Kruskal–Wallis test followed by post hoc Dunn’s test.

Article Snippet: For blocking experiments, TLR4-functionalized cantilevers were exposed to a 100 μg/mL anti-TLR4 monoclonal antibody (MAB14782, R&D Systems) solution in PBS for 1 h. A monoclonal mouse IgG antibody (Human MD-2 Antibody, MAB1787, R&D Systems) was used as negative control.

Techniques: Blocking Assay, Cell Culture, Adhesive, Expressing, Binding Assay, Negative Control, Standard Deviation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: PTX3 binds MD-2 and promotes TRIF-dependent immune protection in aspergillosis.

doi: 10.4049/jimmunol.1400814

Figure Lengend Snippet: FIGURE 1. PTX3 binds to HEK293 cells expressing TLR4 and MD-2. (A) HEK293 cells expressing TLR4/MD-2/CD14, TLR4A, CD14/MD-2, or the empty vector (Null) were incubated with a histidine-tagged PTX3 (25 mg/2 3 105 cells). FACS analysis was performed using an anti-histidine Ab. (B) FACS analysis of HEK293 cells expressing TLR4 alone were incubated with PBS (thick line) or rMD-2 (10 mg/106 cells) (thin line) and sub- sequently with recombinant biotinylated PTX3 (2 mg). PTX3 was detected by FITC–streptavidin. Cells incubated with FITC-streptavidin alone (dotted line). (C) SDS-PAGE analysis of streptavidin beads incubated with biotinylated PTX3, MD-2, and PTX3 with MD-2. The relative positions of the proteins on the gel are indicated by the arrows: MD-2 (MD-2 recombinant protein), biotinylated PTX3 (PTX3), MD-2 plus streptavidin beads (MD-2+beads), PTX3, MD-2, and streptavidin beads (PTX3+MD-2+beads), and PTX3 biotinylated plus streptavidin-beads (PTX3+beads). (D) Immobilized MD-2 was incubated with a 0.4–58 nM range of biotinylated PTX3. Data were analyzed with nonlinear fitting methods. Inset: Scatchard plot of binding data. (E) PTX3-coated plates were incubated with a 2–130 nM range of MD-2 followed by anti–MD-2 Ab, anti-mouse biotinylated Ab, and HRP-STR. (F) MD-2–coated plates (5 mg/ml) were incubated with full-length, N terminus, or C terminus PTX3 followed by polyclonal bio- tinylated anti-PTX3 and HRP–STR. (G) MD-2–coated plates were incubated with biotinylated PTX3 preincubated with MNB4 or MNB1 at the indicated Abs: protein molar ratio followed by HRP–STR. (H) Heat-inactivated conidia of A. fumigatus (5 M/well) were incubated with MD-2 at the indicated concentrations. Bound MD-2 was revealed with an anti–MD-2 polyclonal Ab followed by an HRP-conjugated secondary Ab (n = 10 6 SE). (I) Heat-inactivated conidia were preincubated with MD-2 (at 1 mg/ml), and then PTX3 was applied at the reported titers. Bound PTX3 was assessed with an anti-PTX3 polyclonal Ab and an HRP-conjugated secondary Ab (n = 10 6 SE).

Article Snippet: Following extensive washing with HBS-T, bound proteins were revealed by either a sheep anti-MD-2 polyclonal Ab (R&D Systems) and an HRP-conjugated donkey anti-sheep polyclonal Ab or a rabbit antiPTX3 polyclonal Ab and an HRP-conjugated donkey anti-rabbit secondary Ab.

Techniques: Expressing, Plasmid Preparation, Incubation, Recombinant, SDS Page, Binding Assay