Cxcl1

The following ELISA kits were used for rat tissue: IL-1β, TNF-α, chemokine ligand 1 (CXCL1), IL-6, and α-1-acid glycoprotein (α-1-AGP) (R&D Systems, Minneapolis, Minnesota). The following ELISA kits were used for THP-1 cell culture supernatants: IL-1β (Life Technologies Inc., Burlington, ON) and TNF-α, CXCL1, and IL-6 (R&D Systems, Minneapolis, Minnesota).

The following ELISA kits were used for THP-1 cell culture supernatants: IL-1β, CXCL1, and CRP (R&D Systems, Minneapolis, MN, USA). The following ELISA kits were used for rat tissue: IL-1β, TNF-α, CXCL1, α-1-acid glycoprotein (α1AGP), and calprotectin (S100A8/A9 heterodimer; R&D Systems, Minneapolis, MN, USA), and corticosterone (Invitrogen, Waltham, MA, USA).

Catalytic activities of SpyCEP constructs were measured through detection of remaining intact CXCL8 or CXCL1 substrate following incubation with SpyCEP by ELISA (R&D Systems human CXCL8 and CXCL1 DuoSet ELISA) as per manufacturer’s instructions. For cleavage time courses of CXCL8 by C-terminal SpyCEP, 5 pmol CXCL8 were incubated with C-terminal constructs at a range of enzyme: chemokine molar ratios, 1:5–1:250, in a final volume of 100 μl at room temperature for 60 min. Full-length SpyCEP and C-terminal ^S617A were included as controls at a molar ratio of 1:1000 and 1:5, respectively. To compare SpyCEP cleavage rates of CXCL8 and CXCL1, 5 fmol or 10 fmol SpyCEP were incubated with 2 pmol human CXCL8 and CXCL1 respectively (R&D Systems), in a final volume of 100 μl and incubated at room temperature for 30 min. All reactions were halted at defined timepoints with the addition of concentration of Pefabloc (Sigma-Aldrich) to a final concentration of 2 mg/ml (8.34 mM).
Linear regression analyses of the initial five time points of CXCL1 and CXCL8 cleavage (0, 1, 2, 3 and 4 min) were utilised to determine the maximal rate of SpyCEP activity.