Journal: Scientific reports

Article Title: Structure-activity studies of Streptococcus pyogenes enzyme SpyCEP reveal high affinity for CXCL8 in the SpyCEP C-terminal.

doi: 10.1038/s41598-023-46036-9

Figure Lengend Snippet: Figure 2. Cleavage activity of SpyCEP and C-terminal SpyCEP245–1613 constructs using CXCL8 ELISA. SpyCEP constructs were co-incubated with 5 pmol CXCL8. Graphs show residual CXCL8 after a 60-min room temperature incubation, using full-length SpyCEP at a 1:1000 ratio to CXCL8; the C-terminal SpyCEP construct at a 1:5–1:250 molar ratio to CXCL8; and the C-terminalS617A mutant at a 1:5 molar ratio to CXCL8. Reactions were halted at specified timepoints by the addition of Pefabloc to a final concentration of 2 mg/ml. N = 6 experimental replicates for each construct, data points show means, error bars represent SD. ns p > 0.05, * p ≤ 0.05, **** p ≤ 0.0001, at 60 min as determined by ordinary one-way ANOVA.

Article Snippet: Membranes were subsequently blocked for 1 h at room temperature in blocking buffer (PBS with 5% skimmed milk powder (Sigma-Aldrich) and 0.1% Tween), then blotted overnight at 4 °C with 2 primary antibodies, 1 μg/ml mouse anti-human CXCL8 (R&D Systems) and 1:1000 rabbit anti-ENWVQ.

Techniques: Activity Assay, Construct, Enzyme-linked Immunosorbent Assay, Incubation, Mutagenesis, Concentration Assay

Journal: Scientific reports

Article Title: Structure-activity studies of Streptococcus pyogenes enzyme SpyCEP reveal high affinity for CXCL8 in the SpyCEP C-terminal.

doi: 10.1038/s41598-023-46036-9

Figure Lengend Snippet: Figure 1. Cleavage activity of recombinant SpyCEP constructs assayed by immunoblot. Two colour immunoblot showing cleavage of 18.75 pmol CXCL8 when incubated for 2 h at 37 °C either alone (2nd lane) or with a panel of SpyCEP constructs at a 1:50 molar ratio (SpyCEP: CXCL8). Green bands represent intact CXCL8 (anti-CXCL8 antibody); red bands represent cleaved CXCL8 (anti-ENWVQ). A 17 kDa molecular weight marker is shown in blue, 8 kDa and 6 kDa molecular weights are highlighted by arrows. Figure has been cropped and the original blot is presented in supplementary Fig. S6. Figure is representative of 2 independent immunoblots.

Article Snippet: Membranes were subsequently blocked for 1 h at room temperature in blocking buffer (PBS with 5% skimmed milk powder (Sigma-Aldrich) and 0.1% Tween), then blotted overnight at 4 °C with 2 primary antibodies, 1 μg/ml mouse anti-human CXCL8 (R&D Systems) and 1:1000 rabbit anti-ENWVQ.

Techniques: Activity Assay, Recombinant, Construct, Western Blot, Incubation, Molecular Weight, Marker

Journal: Scientific reports

Article Title: Structure-activity studies of Streptococcus pyogenes enzyme SpyCEP reveal high affinity for CXCL8 in the SpyCEP C-terminal.

doi: 10.1038/s41598-023-46036-9

Figure Lengend Snippet: Figure 4. Kinetic activity of full-length and C-terminal SpyCEP construct cleavage of CXCL8. Data points represent the mean change in cleaved CXCL8 production over time (M s-1) of, (A) Full-length SpyCEP and (B) C-terminal SpyCEP. Error bars represent the standard error of the mean of 5 reactions.

Article Snippet: Membranes were subsequently blocked for 1 h at room temperature in blocking buffer (PBS with 5% skimmed milk powder (Sigma-Aldrich) and 0.1% Tween), then blotted overnight at 4 °C with 2 primary antibodies, 1 μg/ml mouse anti-human CXCL8 (R&D Systems) and 1:1000 rabbit anti-ENWVQ.

Techniques: Activity Assay, Construct

Journal: Scientific reports

Article Title: Structure-activity studies of Streptococcus pyogenes enzyme SpyCEP reveal high affinity for CXCL8 in the SpyCEP C-terminal.

doi: 10.1038/s41598-023-46036-9

Figure Lengend Snippet: Figure 3. Cleavage of CXCL8 and CXCL1 by recombinant full-length SpyCEP. 30-min, room temperature cleavage time course of: (A) 2 pmol CXCL8 with 5 fmol SpyCEP and (B) 2 pmol CXCL1 with 10 fmol SpyCEP. Cleavage reactions were halted at specified timepoints by the addition of Pefabloc to a final concentration of 2 mg/ml and the remaining chemokine measured by ELISA. N = 6 experimental replicates for each data point, error bars represent SD of mean values.

Article Snippet: Membranes were subsequently blocked for 1 h at room temperature in blocking buffer (PBS with 5% skimmed milk powder (Sigma-Aldrich) and 0.1% Tween), then blotted overnight at 4 °C with 2 primary antibodies, 1 μg/ml mouse anti-human CXCL8 (R&D Systems) and 1:1000 rabbit anti-ENWVQ.

Techniques: Recombinant, Concentration Assay, Enzyme-linked Immunosorbent Assay