Materials and methods
Reagents. Chemicals for cell culture were purchased from Invitrogen (Carlsbad, CA) and all other chemicals from Sigma–Aldrich (St. Louis, MO) unless stated otherwise. Antibodies against human IL-1β and against human meprin β were from Santa Cruz Biologicals (Santa Cruz, CA).
Purification of meprin A from rat kidney cortex. Meprin A was purified from the kidney cortices essentially as we described previously [4].
Expression, purification, and analysis of human recombinant meprin β. The meprin β SGC-CBP30 construct, pMepβΔTM1puro3GW, was prepared and analyzed as described in our previous studies [11].
Expression and purification of recombinant proIL-1β. Recombinant proIL-1β was expressed and purified as described in our previous studies [11].
Biological activity assay of processed proIL-1β. pro-IL-1β (163 nM) was digested with 2.8 nM trypsin-activated meprin α, 2.4 nM meprin β, 1.7 nM meprin A, or 8 nM caspase-1 in a endergonic total volume of 100 μl and sterilized using 0.45 μm Millex-GV syringe filters (Millipore). Cell proliferation assays were performed in duplicate in three independent experiments using the helper T-cell line D10S as described [11].
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