The identification of specific peptides against cPLA2-IVA was performed by the screening of a random library of linear dodecapeptides (Ph.D.-12, New England Biolabs Inc., Bioké, Leiden, The Netherlands) against the C2 domain of the cPLA2-IVA (C2-cPLA2-IVA, Recombinant cPLA2, MyBioSource, San Diego, USA), as previously described [45 (link)]. Selection steps were the evaluation of (a) the apparent dissociation constant (K*d) reflecting the affinity of phage clones to the C2-cPLA2-IVA, and (b) the half-maximal inhibitory concentration IC50, reflecting their ability to block the binding of cPLA2-IVA to PC. For the Kd* determination, a range of 10 concentrations for each phage clone was used, reflecting dose-dependence binding to this domain. Complete protocols concerning these specific steps are available in Supplementary Methods .
Selected peptides were synthesized coupled with biotin at their N-terminus (Eurogentec, Seraing, Belgium), allowing their detection. The biotin was spaced from the peptides by a small polyethylene glycol molecule (PEG; 8-amino-3,6-dioxaoctanoic acid). The C-terminus was blocked by amidation.
Selected peptides were synthesized coupled with biotin at their N-terminus (Eurogentec, Seraing, Belgium), allowing their detection. The biotin was spaced from the peptides by a small polyethylene glycol molecule (PEG; 8-amino-3,6-dioxaoctanoic acid). The C-terminus was blocked by amidation.
