Recombinant Protein Cleavage Assay

Recombinant cat S was generated as previously described^{11 (link), 16 (link)} and diluted in 1× PBS, pH 7.4, 5 mM EDTA, 5 mM DTT and 5 mM cysteine chloride. Papain (Sigma-Aldrich) was diluted in 0.1 × PBS, 50 mM sodium acetate, pH 6.5, 5 mM EDTA, 5 mM DTT and 5 mM cysteine chloride. Crystalline Papain is more active and has a broader cleavage range than recombinant cat S. Different incubation times or concentrations were thus used as indicated. N-terminal peptides derived from the sequence of MrgprC11 were obtained from GenScript and diluted in PBS. The 28 amino acid N-terminal peptide MDPTISSHDTESTPLNETGHPNCTPILT was made by Peptide 2.0 (Chantilly, VA). The cysteine protease inhibitor E-64 was obtained from Sigma-Aldrich. Similar results were obtained at either 5 or 10 µM of this compound. Anti-Gaussia luciferase, catalog # E8023S from New England Biolabs was used at a dilution of 1:1500. Anti-phospho-PKC (pan) (βII Ser660) catalog #9371 from Cell Signaling Techology was used at a dilution of 1:1,000. HRP-labeled donkey anti rabbit, catalog # NB660-894 and anti-mouse antibodies catalog # NB7544 were from Thermo Fisher and used at 1:2,000 and 1:5,000 respectively. MW markers were Bioreagents EZ-run prestained rec protein ladder, cat # 3603500 from Thermo Fisher. All other reagents were purchased from Invitrogen unless otherwise noted.